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Synthesis and preclinical evaluation of 18F-PEG3-FPN for the detection of metastatic pigmented melanoma Xiaodong Xu, Lujie Yuan, Lianglan Yin, Yaqun Jiang, Yongkang Gai, Qingyao Liu, Yichun Wang, Yongxue Zhang, and Xiaoli Lan Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00607 • Publication Date (Web): 16 Oct 2017 Downloaded from http://pubs.acs.org on October 23, 2017

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Molecular Pharmaceutics

Synthesis and preclinical evaluation of

18

F-PEG3-FPN for the detection of

metastatic pigmented melanoma

Authors: Xiaodong Xu1,2, Lujie Yuan1,2, Lianglan Yin1,2, Yaqun Jiang1,2, Yongkang Gai1,2, Qingyao Liu1,2, Yichun Wang1,2, Yongxue Zhang1,2*, and Xiaoli Lan1,2*

Affiliations: 1

Department of Nuclear Medicine, Union Hospital, Tongji Medical College,

Huazhong University of Science and Technology, Wuhan 430022, China 2

Hubei Key Laboratory of Molecular Imaging, Union Hospital, Tongji Medical

College, Huazhong University of Science and Technology, Wuhan 430022, China

*Corresponding author: Xiaoli Lan, MD, PhD; Yongxue Zhang, MD Address: Department of Nuclear Medicine, Wuhan Union Hospital, No. 1277 Jiefang Ave, Wuhan, Hubei Province 430022, China. Tel.: +86-27-83692633(O), +86-13886193262 (mobile); Fax: +86-27-85726282. E-mail: [email protected] (X Lan); [email protected] (Y Zhang)

Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 81271623).

Conflict of Interest: The authors declare no potential conflicts of interest.

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Table of Content / Abstract Graphic


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ABSTRACT

18

Although

F-5-fluoro-N-(2-[diethylamino]ethyl)picolinamide

(18F-5-FPN)

is

considered a promising radiopharmaceutical for PET imaging of melanoma, it accumulates at high concentrations in the liver. The aim in this research was to optimize the structure of

18

F-5-FPN with triethylene glycol to reduce liver uptake as

well as improve pharmacokinetics, and to evaluate its performance in detection of melanoma liver and lung metastases. 18F-PEG3-FPN was successfully prepared with a high radiolabeling yield (44.68% ± 5.99%) and radiochemical purity (> 99%). The uptake of

18

F-PEG3-FPN by pigmented B16F10 melanoma cells was significantly

higher than that by amelanotic melanoma A375 cells. The binding to B16F10 cells could be blocked by excess

19

F-PEG3-FPN. On small animal PET images, B16F10

tumors, but not A375 tumors, were clearly delineated after

18

F-PEG3-FPN injection.

More importantly, 18F-PEG3-FPN uptake by liver (2.27 ± 0.45 and 1.74 ± 0.35 %ID/g, at 1 and 2 h) was significantly lower than that of

18

F-5-FPN, and the lesions in lung

and liver could be clearly detected by 18F-PEG3-FPN PET imaging in mouse models of pulmonary or hepatic metastases. Overall, we successfully synthesized 18

F-PEG3-FPN,

which

has

higher

labeling

efficacy

and

pharmacokinetics along with lower liver uptake compared to suggests

18

better 18

in

vivo

F-5-FPN. This

F-PEG3-FPN as a candidate for pigmented melanoma liver and lung

metastasis detection.

Keywords: melanoma; benzamides; PET; metastasis; pharmacokinetic



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INTRODUCTION

Malignant melanoma accounts for 90% of cutaneous tumor deaths, and exhibits a high potential for metastatic spread [1]. Early melanoma (stages I and II) is highly localized and can be treated by surgery and adjuvant treatment. However, once it reaches stage III local metastasis or stage IV distant metastasis, the prognosis often becomes poor and the survival rate significantly drops. There are major differences in survival rate according to the localization of metastasis. For example, the 5-year survival rates of patients with only cutaneous metastases is 18.8%, when the melanoma spreads to lung and other organs, it drops to 6.7%–9.5% [2]. Therefore, earlier detection and assessment of metastasis may be helpful for increasing the survival rate through accurate staging and more appropriate therapeutic interventions.

In clinic practice, positron emission tomography (PET) plays an increasingly critical role

on

the

diagnosis

and

evaluation

of

metastatic

melanoma.

[18F]-fluorodeoxyglucose (18F-FDG) is broadly applied for tumor PET imaging and staging. However,

18

F-FDG is nonspecific [3], and lacks enough sensitivity for PET

imaging of sentinel lymph node metastases in stages I and II melanoma [4,5]. Furthermore,

18

F-FDG PET/CT fails to highlight micrometastases ( 90% of primary melanoma cases [13,14], is an extremely promising target for molecular imaging and therapy of melanoma. In the last few years, many melanin-targeted radiopharmaceuticals based on benzamide, quinoxaline, or picolinamide have been developed as PET and SPECT agents, such as [15],

123

I-MEL008 [16], 4-11C-MBZA [17] and

18

18

F-FBZA

F-MEL050 [18,19]. More recently,


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our 18

research

group

also

successfully

synthesized

a

PET

tracer,

F-5-fluoro-N-(2-[diethylamino]ethyl)picolinamide (18F-5-FPN), which exhibits high

stability, tight binding to melanin, and high specificity [20]. Unfortunately, like most benzamide/nicotinamide-based probes, this probe still shows relatively high uptake in the liver (6.73 ± 1.36 and 5.33 ± 0.94 % ID/g at 1 and 2 h after injection) [20], which decreases the sensitivity for the diagnosis of liver metastases. Accordingly, it was necessary to conduct further modification to minimize nonspecific liver uptake.

To this end, based on the precursor of 18F-5-FPN, we designed a method to prepare a 18

F-fluoroPEGylated radiotracer for disseminated melanoma detection. We modified

the 3-position of pyridine (Figure 1) with a short chain polyethylene glycol and then labeled 18

18

with

F

to

produce

F-N-(2-diethylaminoethyl)-4-(2-[2-{2-fluoroethoxy}ethoxy]ethoxy)

(18F-PEG3-FPN). We performed biological characterization of

18

pyridine F-PEG3-FPN in

C57BL/6 mice bearing subcutaneous melanin-positive B16F10 murine melanoma and in BALB/c nude mice bearing A375 human amelanotic melanoma. We assessed its potential diagnostic value for detecting melanoma metastases in murine models of lung metastases and hepatic metastases.

MATERIALS AND METHODS Reagents and Instruments

All

chemicals

were

obtained

from

J&K

Chemicals

(Beijing,

China),

Adamas Reagent Co., Ltd., (Shanghai, China), or Sigma-Aldrich (St. Louis MO, USA) and were used without further purification. Thin layer chromatography (TLC) was performed on silica gel F254 aluminum-backed plates (Qingdao Haiyang Chemical Co., Ltd., China) with visualization under UV (254 nm). NMR spectra were recorded on a Bruker 400 MHz spectrometer (Bruker, Karlsruhe, Germany). The mass spectrometry (MS) spectra were recorded on a Thermo LCQ DECA XP plus ESI-MS (Thermo Fisher, Palo Alto, CA, USA). Analytic and semipreparative high performance liquid chromatography (HPLC) was carried out on a system (LC-10AT,



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Shimadzu Corporation, Tokyo, Japan) equipped with an SPD-10A UV/VIS detector (Shimadzu) and a flow count radiation detector (Bioscan, Washington DC, USA) for gamma ray detection. Radioactivity was quantified using a dose calibrator (CRC-15R, Capintec, Ramsey NJ, USA) or a γ-scintillation counter (2470 WIZARD; PerkinElmer, Waltham MA, USA). Radiochemical purity and stability of the final product was analyzed by HPLC using a C-18 column (4.6 × 250 mm, 5 µ), eluted at a flow rate of 1.0 mL/min. The mobile phase started from 5% MeCN/water containing 0.1% trifluoroacetic acid (TFA) at 0–5 min, then ramped to 60% MeCN/water at 35 min.

Synthesis of 5-(2-[2-{2-hydroxy-ethoxy}-ethoxy]-ethoxy)-pyridine-2-carboxylic acid (2-diethylamino-ethyl)-amide (2)

571 mg of 70% sodium hydride in paraffin oil (400 mg of NaH, 17 mmol) was added to a solution of triethylene glycol (3 g, 20 mmol) in 20 mL of N,N Dimethylformamide (DMF). Compound 1 (1 g, 3.33 mmol) was added [21]. After stirring at 100°C for 20 h, DMF was removed under vacuum and the residue was partitioned between 100 mL water and 50 mL dichloromethane (DCM). The aqueous layer was extracted 3 times with DCM. The combined organic layers were dried over Na2SO4. The crude mixture was purified by silica gel chromatography (DCM/MeOH 10:1) to obtain Compound 2 as a yellowish oil (670 mg, 54% yield). 1H NMR (400 MHz, MeOD) revealed δ 8.30 (d, J = 2.6 Hz, 1H), 8.02 (d, J = 8.7 Hz, 1H), 7.48 (dd, J = 8.7, 2.9 Hz, 1H), 4.26 (dd, J = 5.3, 3.7 Hz, 2H), 3.94–3.81 (m, 2H), 3.71 (td, J = 3.8, 1.1 Hz, 2H), 3.67–3.61 (m, 4H), 3.57–3.48 (m, 4H), 3.30 (dt, J = 3.3, 1.6 Hz, 2H), 2.75 (t, J = 6.9 Hz, 2H), 2.68 (q, J = 7.2 Hz, 4H), 1.09 (t, J = 7.2 Hz, 6H). 13C NMR (101 MHz, MeOD) δ 165.44, 157.60, 142.12, 137.20, 122.89, 120.74, 72.31, 70.42, 70.05, 69.19, 67.95, 60.82, 51.25, 48.25, 48.03, 47.82, 47.61, 47.40, 47.18, 46.97, 46.89, 36.29, 10.13. HRMS m/z (ESI+)：calcd for C18H31N3O5H[M + H]+ 370.2336, found 370.2388.

Synthesis

of

Toluene-4-sulfonic

acid

2-(2-[2-{6-(2-diethylamino-ethylcarbamoyl)-pyridin-3-yloxy}-ethoxy]-ethoxy)-


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ethyl ester (3)

A solution of paratoluensulfonyl chloride (1.2 g, 8.12 mmol) in 10 mL anhydrous pyridine was cooled to 0°C, Compound 2 (1 g, 2.71 mmol) was added. After stirring at room temperature for 4 h, Pyridine was removed under reduced pressure. The mixture was extracted with DCM (100 mL) and the organic layers was washed with water (50 mL × 3). The organic phase was dried over Na2SO4. The crude mixture was purified by silica gel chromatography (DCM /MeOH 10:1) to obtain compound 3 (795 mg, 56% yield). 1H NMR (400 MHz, CDCl3) revealed δ 8.20 (t, J = 5.3 Hz, 1H), 8.12 (d, J = 2.3 Hz, 1H), 7.99 (d, J = 8.7 Hz, 1H), 7.69 (d, J = 8.0 Hz, 2H), 7.23–7.15 (m, 2H), 4.12–4.07 (m, 2H), 4.08–4.02 (m, 2H), 3.78–3.72 (m, 2H), 3.62–3.54 (m, 4H), 3.51 (dd, J = 6.7, 5.8 Hz, 4H), 2.73 (t, J = 6.4 Hz, 2H), 2.65 (q, J = 7.1 Hz, 4H), 2.33 (s, 3H), 1.03 (t, J = 7.2 Hz, 6H).13C NMR (101 MHz, CDCl3) δ 164.67, 157.11, 144.87, 142.69, 136.90, 132.93, 129.84, 127.92, 123.19, 121.03, 70.77, 69.50, 69.24, 68.73, 68.02, 51.55, 47.31, 36.52, 21.62, 10.89. HRMS m/z (ESI+): calcd for C25H37N3O7SH[M + H]+ 524.2425, found 524.2438. Synthesis

of

5-(2-[2-{2-Fluoro-ethoxy}-ethoxy]-ethoxy)-pyridine-2-carboxylic

acid (2-diethylamino-ethyl)-amide (19F-PEG3-FPN)

Tetrabutylammonium fluoride (TBAF) solution in THF (1 M, 0.4 mL) was added to a solution of compound 3 (105 mg, 0.2 mmol) in tetrahydrofuran (THF) (4 mL). The mixture was refluxed for 12 h at 70°C. THF was removed. The residue was purified using silica gel column chromatography eluting with DCM/MeOH (10:1) afforded 19

F-PEG3-FPN (36 mg, 48% yield). 1H NMR (400 MHz, CDCl3) revealed δ 8.29 (d, J

= 2.7 Hz, 1H), 8.16 (d, J = 8.7 Hz, 1H), 7.34 (dd, J = 8.7, 2.9 Hz, 1H), 4.72–4.64 (m, 1H), 4.60–4.47 (m, 1H), 4.31–4.22 (m, 2H), 4.01–3.92 (m, 2H), 3.85–3.82 (m, 1H), 3.81–3.78 (m, 2H), 3.76 (dd, J = 5.9, 2.9 Hz, 3H), 3.60 (q, J = 6.3 Hz, 2H), 2.78 (t, J = 6.4 Hz, 2H), 2.70 (q, J = 7.1 Hz, 4H), 1.13 (t, J = 7.1 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 164.45, 157.05, 142.95, 136.88, 123.20, 120.97, 83.98, 82.30, 77.37, 77.26, 77.05, 76.74, 70.95, 70.86, 70.58, 70.38, 69.57, 68.01, 51.70, 47.18, 37.01, 11.54. HRMS m/z (ESI+): calcd for C19H3119FN2O4H[M + H]+ 371.2341, found 372.2309.



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Radiochemical

synthesis

5-(2-[2-{2-18Fluoro-ethoxy}-ethoxy]-ethoxy)-pyridine-2-carboxylic

Page 8 of 32

of acid

(2-diethylamino-ethyl)-amide (18F-PEG3-FPN)

All processes were performed in the GE Tracerlab FXFN. Aqueous [18F] fluoride solution was transferred into a target vial under nitrogen gas pressure and then transferred through a quarternary methylamine (QMA) cartridge (Sep-Pak, Waters, Inc. Milford MA, USA). The [18F] fluoride trapped on the cartridge was eluted to the reactor with an elution of K2,2,2 (15 mg in 1 mL CH3CN) and K2CO3 (3 mg in 0.5 mL H2O). The K[18F]/K2,2,2 solution in the reactor was evaporated at 85°C under reduced-pressure nitrogen flow for 5 min. To the dry residue we added the solution of Compound 3 (5 mg in anhydrous 0.6 mL DMSO) and the mixture was heated at 100°C for 10 min, then cooled down to 35°C. The crude product was dissolved with MeCN, then injected into the C18 semipreparative HPLC column. The mobile phase started from 20% MeCN/water containing 0.1% TFA, then changed to 50% MeCN/water at 5 min with a flow rate of 6 mL/min. The desired fractions were combined, evaporated under reduced pressure, redissolved in normal saline, and filtered through a 0.22 µm aseptic membrane filter to afford the final product for in vitro and in vivo studies.

Partition Coefficient 18

F-PEG3-FPN (74 kBq, 20 µCi) was added to a mixed solution of phosphate-buffered

saline (PBS) (pH 7.4, 1.0 mL) and 1-octanol (1.0 mL) in a 5 mL centrifuge tube. The mixed solution was vigorously vortexed for 5 min and centrifuged (3000 rpm, 5 min), a sample (100 µL) from each phase was quantified for radioactivity using a γ-counter. The partition coefficient was expressed as Log P = Log10 (counts in 1-octanol/counts in PBS).

In Vitro Serum Stability of 18F-PEG3-FPN 18

F-PEG3-FPN (3.7 MBq, 100 µCi) was added into human serum (0.5 mL) and


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incubated at 37 °C. Aliquots (200 µL) of serum was sampled at 1, 3 and 6 h. Plasma protein was precipitated with 0.5 mL acetonitrile and centrifuged (13000 rpm, 5 min). The radiochemical purities of

18

F-PEG3-FPN in filtrates were assayed by analytic

HPLC under analytic conditions as described above.

In Vitro Cell Study

Pigmented melanoma B16F10 and amelanotic melanoma A375 cells were cultured in Dulbecco’s modified Eagle high-glucose medium (DMEM; Gibco, Carlsbad CA, USA) containing 10% fetal bovine serum (Gibco) and antibiotics (100 mg/mL streptomycin and 100 mg/mL penicillin; Gibco) at 37°C in a humidified incubator with 5% CO2.

B16F10 and A375 cells were grown in flasks to 80%–90% confluence and were used for cell uptake studies. Sets of four 24-well plates containing 1 × 105 cells/well were incubated with 18F-PEG3-FPN (0.5 pM, 2 µCi) for 30, 60 and 120 min at 37°C. After removing the medium, the cells were washed twice with ice-cold PBS, pH 7.4. The adherent cells were then washed twice with glycine and lysed with 1 N NaOH at 37°C for 10 min. The combined washes and lysate were measured with a γ-counter.

Next, the blocking study was conducted in B16F10 cells. The cells were incubated for 1 h at 37°C with 18F-PEG3-FPN (0.5 pM, 2 µCi), with or without the pretreatment of 100 µL excess standard

19

F-PEG3-FPN (500 pM). After removing the medium, the

cells were washed twice with ice-cold PBS. The cells were then washed twice with glycine and lysed with 1 N NaOH at 37°C for 10 min. The combined washes and lysate was measured with a γ-counter.

Animal Models

All animal studies were performed according to the guidelines of the Institutional Animal Care and Use Committee of Tongji Medical College of Huazhong University of Science and Technology. C57BL/6 male mice (4–6 weeks old) and male



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BALB/C-nu/nu mice (4–6 weeks old) (Beijing Vital River Laboratory Animal Technology Co., Ltd., Beijing, China) were kept under sterile conditions. For the primary subcutaneous melanoma model, C57BL/6 mice were inoculated in the right shoulder with B16F10 cells (1 × 105 in 100 µL PBS), and BALB/C-nu/nu mice were injected A375 cells (2 × 106 in 100 µL PBS) into the right shoulder. To mimic the melanoma pulmonary metastases, B16F10 cells in 200 µL PBS were injected into C57BL/6 mice via tail vein at a concentration of 62500 cells/mL or 12500 cells/mL [22]. Melanoma hepatic metastases models were prepared by injecting the B16F10 cells (2 × 106 in 100 µL PBS) slowly into the exposed hemispleen [23].

Small-animal PET Scanner Imaging

All animals were anaesthetized with 50 mg/kg pentobarbital and maintained with 2% isoflurane in 100% oxygen during the imaging process. Tumor-to-background ratio (TBR) and lesion-to-background ratio (LBR) were calculated using a medical imaging data analysis software (Amide.exe 1.0.4).

As our previously literature detailed [20], C57BL/6 mice bearing B16F10 tumors (n=5) were attached to the center of scanner (BioCaliburn LH Raycan Technology Co., Ltd., Suzhou, China) in a prone position, then

18

F-PEG3-FPN (3.7 MBq) was given

through the tail vein and 40-min dynamic scans were obtained (1 q 10 s × 6, 1 q 30 s × 4, 1 q 60 s × 4, 1 q 2 min × 4, 1 q 5 min × 3, 1 at 10 min, total of 22 frames). Similarly, mice bearing B16F10 (n=5) or bearing A375 (n=5) subcutaneous melanoma were administered with

18

F-PEG3-FPN (3.7 MBq), and static images (10 min) were

acquired at 1 h post injection (pi). In the blocking group, images (10 min) were obtained approximately 1 h pi of

18

F-PEG3-FPN (3.7 MBq) and

19

F-PEG3-FPN (500

µg) into five mice bearing B16F10 subcutaneous melanoma. To further assess the ability of 18F-PEG3-FPN to detect metastatic pigmented melanoma, the C57BL/6 mice with pulmonary and hepatic metastasis models were scanned 1 h after injection of 18

F-PEG3-FPN via the tail vein.


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Tissue biodistribution Studies

The mice (B16F10 and A375, n = 5 in each group) were euthanized 1 and 2 h pi of 18

F-PEG3-FPN (3.7 Mq), the organs and tumor were removed, weighed and

radioactivity was counted using a γ-counter. The radioactivity in each organ was normalized as the percentage of injected dose per gram of tissue (%ID/g). In the blocking group, the same experiment was undertaken in the B16F10 mice (n = 5) 1 h pi of 18F-PEG3-FPN (3.7 MBq) and 500 µg 19F-PEG3-FPN.

Histopathology Examination

The collected lung and liver samples were fixed in 4% paraformaldehyde, and embedded in paraffin. These samples were cut into 4-µm-thick sections, deparaffnized in xylene and serially dehydrated in decreasing concentrations of ethanol. Then the sections were stained with hematoxylin and eosin (H&E) and examined under a light microscope.

Statistical Analysis

The quantitative data are reported as mean ± SD. Statistical analysis was performed by one-way ANOVA or Student’s t-test with GraphPad Prism (version 5.01). P < 0.05 was considered to have statistical difference.

RESULTS Chemical and Radiochemical 18

F-PEG3-FPN was successfully prepared in a straightforward manner using a

three-step procedure (Figure 1). After purification by semipreparative HPLC, the final product

18

F-PEG3-FPN was obtained with a radiochemical yield of 44.68% ± 5.99%

(n = 4). The radiochemical purity is more than 99% (Figure 2A). The specific activity was determined as 120–195 GBq/µmol based on the measurement of radioactivity and the 18F-PEG3-FPN concentration via radio-HPLC. The overall time for synthesis was approximately 45 min, including the process of purification and reformulation. The identity of

18

F-PEG3-FPN was conducted by co-injection with a non-radioactive



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standard 19F-PEG3-FPN (Figure 2A).

Characterization of 18F-PEG3-FPN

The stability results of

18

F-PEG3-FPN in human serum are shown in Figure 2 B–D.

The percentage of intact 18F-PEG3-FPN was > 99% after incudbation for 3 and 6 h at 37°C. Defluorination was not observed in human serum, even after 6 h incubation. A Log P of − 0.69 ± 0.02 revealed that

18

F-PEG3-FPN is more hydrophilic than

unPEGylated 18F-5-FPN (Log P =﹣0.17 ± 0.03).

In Vitro Cell Study

Uptake studies of 18

18

F-PEG3-FPN were conducted to assess the specific binding of

F-PEG3-FPN toward melanin-expressing tumor cells. As Figure 3A shows, the

radioactivity level of

18

F-PEG3-FPN in B16F10 cells increased in a time-dependent

fashion. Instead, the uptake in A375 cells were significantly lower. In another group of B16F10 cells pre-treated with a blocking dose of 18

19

F-PEG3-FPN, the

F-PEG3-FPN uptake was significantly reduced by approximately 20-fold, indicating

high specificity for melanin.

Small animal PET Imaging Studies

The dynamic small animal PET scans and time-activity curves (Figure 4A and B) show rapid clearance of the tracer from the urinary system. At 20 s pi of 18

F-PEG3-FPN, the tracer rapidly concentrated in the kidneys (35.28 ± 7.422 %ID/g).

By 40 min, it had decreased to 5.54 ± 0.89 %ID/g. Similarly, the uptake in liver gradually decreased from 12.23 ± 2.41%ID/g at 20 s pi to 2.84 ± 0.55%ID/g at 40 min. Twenty min after injection, lung uptake sharply decreased to 2.37 ± 0.12%ID/g, approaching the uptake of muscle. The activity level in thyroid also changed in a similar fashion. In contrast, tumor uptake immediately became visible at 1 min (6.59 ± 1.17%ID/g) after tracer injection, indicating rapid binding, and peaked (18.56 ± 1.05%ID/g) at 40 min pi.


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The static images obtained 1h pi of

18

F-PEG3-FPN (Figure 5A) displayed excellent

tumor retention and high tumor-to-background ratio in mice bearing B16F10 tumor, whereas no tumor uptake was detected in mice bearing the amelanotic A375 melanoma (Figure 5C). In addition, the blocking study in mice bearing B16F10 tumor showed the tumor was still visible but the tumor uptake dramatic decreased (Figure 5B).

In view of the excellent imaging performance of 18F-PEG3-FPN in lung and liver, we assessed its potential diagnostic value for detecting melanoma hepatic and lung metastatic lesions (Figure 6 and Figure 7). In the hepatic metastatic model, two liver metastases were clearly visible on the 7-day images, with lesion-to-background ratios of 8.36 and 8.43 (Figure 6A). In another mouse, the lesion-to-background ratio in diffuse hepatic metastases reached up to 15.90 (Figure 6B).

18

F-PEG3-FPN PET

scanning clearly delineated the pulmonary metastases (< 2 mm) of B16F10 melanoma in C57 mice with high lesion-to-background ratios of 8.39 and 8.87 (Figure 7A). The liver and lung lesions and diffuse tumor burden were also clearly visible in the tissue specimens (Figure 6C, 6D, 7C and 7D). For the histopathology examination, H&E staining proved the lesions in lung and liver were metastatic melanoma (Figure 6E and 7E).

Tissue biodistribution studies

The biodistribution studies shows (Table 1) the tumor uptake of

18

F-PEG3-FPN was

very high in the B16F10 tumor-bearing mice, with 19.52 ± 1.69 %ID/g at 1 h pi. Furthermore, prolonged retention (19.51 ± 1.51 %ID/g) in tumor was observed at 2 h. While relative low radioactivity levels in the muscle, liver, lung, and blood led to higher TBR. In contrast, amelanotic A375 tumor uptakes were only 2.78 ± 0.49 and 2.35 ± 0.15 %ID/g at 1 and 2 h pi, respectively. As expected, the eyes of C57BL/6 had the highest radioactivity with 25.87 ± 2.55 and 25.22 ± 1.24 %ID/g at 1 h and 2 h, respectively. In contrast, the eyes of BALB/c nude mice showed relatively low radioactivity (3.14 ± 0.39 and 2.84 ± 0.17 %ID/g at 1 h and 2 h, respectively). The



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Page 14 of 32

radioactivity levels in these organs were correlated with to the corresponding PET imaging. Additionally, compared to the unblocked mice, much lower tumor uptake for 18

F-PEG3-FPN in the blocked mice were noted (2.54 ± 0.44 %ID/g).

DISCUSSION

In our experimental design, we aimed to introduce a short PEG moiety to the 5-bromo-N-(2-[diethylamino]ethyl)-2 pyridinecarboxamide core to improve the in vivo pharmacokinetics.

18

F-PEG3-FPN was successfully prepared through a

fluoro-for-tosyl exchange reaction, and the tosyl group in the triethylene glycol side chain is a good leaving group, which makes the fluorization reaction more easily and efficiently. In contrast,

18

F-5-FPN was synthesized through a less efficient

bromine-for-fluorine exchange reaction in the 3-position of pyridine ring, resulting in a lower radiochemical yield. Therefore, the radiolabeling yield of 18F-PEG3-FPN was much higher than that of 18F-5-FPN. The high and reliable yield makes it possible to clinical application.

The dynamic and static images in in B16F10 melanoma and A375 melanoma models showed excellent melanin targeting properties and specificity of

18

F-PEG3-FPN. As

seen in Figure 4A, like 18F-5-FPN, the tumor rapidly became visible only 60 s pi, and the radioactivity level in the tumor continued to increase over 40 min. Conversely, uptake in nontarget organs such as lung and liver displayed fast washout. Interestingly, as previously reported [18,19], there was also high uptake in the thyroid during early dynamic imaging. This uptake had washed out on subsequent static imaging at 1 h and 2 h. This illustrates 18F-PEG3-FPN was quickly cleared from thyroid and will not have an impact on imaging for cervical lymph node metastases. The exact mechanism of 18F-PEG3-FPN uptake by the thyroid is unknown; further research is necessary.

From biodistribution experiments, 1 h after intravenous injection of

18

F-PEG3-FPN,

high uptake in B16F10 melanoma (19.52 ± 1.69 %ID/g) was observed, and it remained stable until 2 h pi (19.51 ± 1.51 %ID/g). Although the uptake of


Page 15 of 32

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18

F-PEG3-FPN by the muscle (1.51 ± 0.29 %ID/g) at 2 h was higher than that of

18

F-5-FPN (0.55 ± 0.08 %ID/g) [20], the sustained highly radioactivity concentration

in tumor led to favorable tumor-to-muscle ratios reaching 13.17 ± 2.09. In addition, due to the higher uveal melanin content in C57BL/6 mice, the radioactivity level in the eyes was 8-9 times higher than that in amelanotic BALB/c nude mice with pink eyes. This further confirmed that Fortunately, compared to

18

18

F-PEG3-FPN specifically binds to melanin.

F-5-FPN, liver showed significantly low uptake of

18

F-PEG3-FPN (P < 0.001). This result is encouraging and demonstrates that

18

F-PEG3-FPN may have a great advantage for the diagnosing of melanoma liver

metastasis. A detail comparison of 18F-PEG3-FPN and 18F-5-FPN was listed on Table 2.

Therefore, we further assessed the potential of 18F-PEG3-FPN as a PET tracer for the diagnosis of metastatic melanoma lesions. We successfully established a mouse model of liver metastasis using the B16F10 cell line in C57BL/6 mice. The pathology showed many cells with enlarged or multiple nuclei and dark brown granules, consistent with melanoma. As shown in Figure 6A, small animal PET scanning detected two liver metastases clearly. These results demonstrate that PET imaging of 18

F-PEG3-FPN has the capability of differentiating melanoma hepatic metastasis from

normal liver tissue.

Hepatic metastasis imaging is still challenging. Various imaging modalities have been applied for assessment of hepatic metastasis, including ultrasound, CT, PET, and MR [24–26]. Currently, PET/CT imaging with 18F-FDG is a critical tool for the staging of malignant melanoma. Nevertheless, the effectiveness of detection of liver metastases from melanoma remains controversial [25,26], as the high radioactivity of normal hepatic tissue often affects the detection of smaller metastases. 18F-PEG3-FPN, with a different mode of action from

18

F-FDG, may be an effective supplement for the

staging of malignant melanoma.



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Page 16 of 32

In addition to liver, lung is another common site of metastasis from melanoma. Similarly, the diffuse lesions in lungs show significantly high radioactivity levels, the lesion-to-background ratio is 13.36. More specifically, two micrometastases (< 2 mm) were observed very clearly, the lesion-to-background ratios is 8.39 and 8.87, respectively.

18

F-5-FPN also can image lung micrometastases (1–2 mm), but with a

lower lesion-to-background ratio [20]. Compared with

18

F-FDG,

18

F-5-FPN PET

could obviously detect metastatic lesions in the pulmonary metastasis model, while in which nearly no lung uptake of

18

F-FDG was noticed [27]. As seen above, these

results confirmed that 18F-PEG3-FPN is an excellent tracer for the early detection of melanoma pulmonary metastases.

In this study, three issues should be noted. First, PET images showed significant higher uptake of

18

F-PEG3-FPN in the diffused liver and lung metastases than in

single metastatic nodule or non-diffused lesions. This may be related to the different levels of tumor burden [19], with higher tumor burden in the diffused metastatic lesions. We speculated a strong correlation between the uptake level of 18F-PEG3-FPN and the tumor burden, but further study should be performed. Second, the uptake in blood and muscle of

18

F-PEG3-FPN was higher than that of

18

F-5-FPN. A possible

explanation for this is the effect of PEGylation for prolonging the circulation time and increasing hydrophilicity (Log P＝− 0.69 vs − 0.17 for

18

F-5-FPN). However, high

tumor-to-muscle ratio (13.17 ± 2.09 at 2 h pi) and tumor-to-blood ratio (10.02 ± 1.73 at 2 h pi) suggest the reduced lipophilicity does not have an appreciably effect on the binding affinity of

18

F-PEG3-FPN to melanin. In other words, it shows that Log P

does not correctly predict the transporting ability of the radiotracer across the cell membrane and the binding capacity to melanin. Third, the intestinal tract is another nontarget tissue with high radioactivity in accordance with previously reported compounds [28,29]. It is probable that the PEG side chain enhanced excretion of this probe via the hepatobiliary system. Unfortunately, this may render metastatic intestinal melanoma undetectable. Last but not the least, the high specificity and


Page 17 of 32

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sensitivity of

18

F-PEG3-FPN for micrometastases along with a lower liver uptake

indicates that this structure also has high potential as a therapeutic agent for the metastatic melanoma once labeling with therapeutic radioisotopes, such as

131

I.

Experiments focused on the radiotherapeutic strategy is going on in our group.

CONCLUSION 18

F-PEG3-FPN can be easily synthesized in high radiochemical yield.

18

F-PEG3-FPN

exhibited high affinity and specificity for high melanin-expressing melanoma. Compared with

18

F-5-FPN,

18

F-PEG3-FPN had better biodistribution for the lower

uptake in the liver, which could detect liver metastasis from melanoma with a higher tumor-to-normal liver ratio. These preclinical results support the potential of 18

F-PEG3-FPN as an excellent PET tracer for detecting melanoma and metastatic

lesions.

ACKNOWLEDGMENTS: This work was supported by the National Natural

Science Foundation of China (No. 81371626). We thank Prof. Xiang Ma, Dr. Shasha Zhang and Dr. Kongchao Wang from the School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, for their technical support in preparation of compounds. We thank Dr. Shuangxue Han from the College of Life Science, Huazhong University of Science and Technology, for her kind help in small animal PET imaging.

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Page 22 of 32

Tables Table 1. Biodistribution of

18

F-PEG3-FPN in difference mice 1 h pi. Values are

expressed as means ± SD (% ID/g). B16F10 melanoma *

A375 melanoma **

Blocked ***

(n=5)

(n=5)

(n=5)

Organ 1h

2h

1h

2h

1h

Blood

3.15±0.59

2.00 ±0.40

2.96 ±0.15

1.66 ±0.36

0.99 ±0.09

Brain

1.81±0.29

1.36 ±0.64

1.67 ±0.22

1.31 ±0.13

0.89 ±0.16

Heart

3.50±0.57

1.82 ±0.51

2.63 ±0.10

1.96 ±0.42

0.77 ±0.32

Lungs

2.45±0.40

1.98 ±0.79

2.08 ±0.52

1.51 ±0.40

1.21 ±0.08

Liver

2.27±0.45

1.74 ±0.35

2.85 ±0.16

1.76 ±0.16

0.92 ±0.11

Spleen

3.74±0.69

2.32 ±1.10

2.26 ±0.13

1.68 ±0.22

1.08 ±0.26

Kidney

4.26±0.38

2.50 ±0.76

2.95 ±0.32

1.84 ±0.15

1.69 ±0.23

Stomach

3.25±0.63

2.40 ±0.80

1.46 ±0.21

1.26 ±0.45

1.17 ±0.32

Large intestine

5.08±0.85

3.98 ±1.00

3.50 ±0.29

2.68 ±0.42

1.05 ±0.25

Small intestine

5.14±0.30

2.23 ±0.57

2.66 ±0.24

1.52 ±0.25

1.32 ±0.36

Muscle

1.75±0.16

1.51 ±0.29

1.49 ±0.31

1.42 ±0.02

0.72 ±0.18

Bone

2.77±0.38

2.61 ±0.36

2.39 ±0.25

2.11 ±0.10

0.41 ±0.05

Tumor

19.52±1.69

19.51 ±1.51

2.78 ±0.49

2.35 ±0.15

2.54 ±0.44

Eyes

25.87±2.55

25.22±1.24

3.14 ±0.39

2.84 ±0.17

2.99 ±0.51

Tumor-to-blood

6.35 ±1.24

10.02 ±1.73

0.94 ±0.20

1.48 ±0.40

2.87±0.30

Tumor-to-liver

8.86 ±1.88

11.58 ±2.47

0.98 ±0.22

1.33 ±0.06

3.29±0.64

Tumor-to-kidneys

4.60 ±0.44

8.56 ±3.07

0.10 ±0.02

0.08 ±0.01

1.77±0.18

Tumor-to-lung

8.12 ±1.37

11.06 ±4.18

1.40 ±0.47

1.64 ±0.43

2.47±0.29

Tumor-to-muscle

11.23 ±1.64

13.17 ±2.09

1.95 ±0.61

1.66 ±0.12

4.26±0.64

Uptake ratio

* B16F10 Melanoma: C57BL/6 mice bearing B16F10 melanoma; **A375 melanoma: BALB/C-nu/nu bearing A375 melanoma; ***Blocked: C57BL/6 mice bearing B16F10 melanoma 1 h pi of both 19

F-PEG3-FPN


18

F-PEG3-FPN and

Page 23 of 32

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Table 2．Comparison of 18F-PEG3-FPN and 18F-5-FPN 18

F-PEG3-FPN

18

F-5-FPN

Chemical structure

Radiolabeling yields Radiochemical pure

44.68% ± 5.99% > 99%

5%–10% > 95%

Specific activities

120–195 GBq/µmmol

100–120 GBq/µmmol

Stability in vitro

good

good

﹣0.69 ± 0.02

﹣0.17 ± 0.01**

Tumor uptake (%ID/g) *

19.51±1.51

16.63 ± 5.41

Liver (%ID/g) *

1.74±0.35

5.33 ± 0.94

Log P

*The uptake values were measured 2 h pi of the tracers in B16F10 tumor-bearing mice. **From our unpublished data.



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Page 24 of 32

Figure Legends

Figure 1 Reaction schemes to synthesize tosyl precursor and standard 19F-PEG3-FPN.

Reagents and conditions: (i) Triethylene glycol, NaH, DMF, 100°C, 20 h; (ii) p-toluensulfonyl chloride, pyridine, room temprature, 4 h; (iii) TBAF, THF, 70°C, overnight; (iv) 18F-KF, K2,2,2 complex, DMSO, 100°C, 10 min.

Figure 2 Identification and in vitro stability study of

18

F-PEG3-FPN. (A) Analytic

HPLC chromatograms after the co-injection of 18F-PEG3-FPN (red, retention time is 21.4 min) and standard unlabeled

19

F-PEG3-FPN (blue, UV 254 nm). (B)

18

F-PEG3-FPN stability in phosphate-buffered saline for 6 h. (C) and (D)

18

F-PEG3-FPN stability in human serum after incubation at 37°C for 3 and 6 h,

respectively.

Figure 3 In vitro cell study. (A) In vitro studies of 18F-PEG3-FPN uptake by B16F10

melanoma cells and A375 amelanotic melanoma cells at 30 min, 60 min, 120 min. The differences in radioactivity over time in A375 cells are statistically insignificant (P > 0.1). (B) Blocking assay of

18

F-PEG3-FPN by

19

F-PEG3-FPN in B16F10

melanoma cells (P < 0.0001).

Figure 4 Dynamic PET imaging. (A) A series of coronal dynamic PET images

obtained from mice bearing B16F10 melanoma tumors. (B) Time-activity curves of organs and tumors.

Figure 5 Static PET imaging of

18

F-PEG3-FPN in primary subcutaneous melanoma

model (1 h pi). Red arrows indicate tumors. Representative images of C57BL/6 mouse bearing B16F10 tumor xenograft (A), B16F10 tumor-bearing mouse blocked with unlabeled 19F-PEG3-FPN (B) and BALB/c mouse bearing A375 tumor xenograft.


Page 25 of 32

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Figure 6 Small animal PET imaging and tissue histologic changes of hepatic

metastasis mouse model bearing B16F10 melanoma cells. (A) Representative coronal 18

F-PEG3-FPN small animal PET images of liver metastatic lesions in liver (red

arrows), with several black nodules < 0.5 cm were discovered in the liver tissue (C, red arrows). (B) Another mouse showed diffuse liver uptake on the PET imaging, with diffused lesions were seen in liver tissue samples (D). The pathology showed many cells with enlarged or multiple nuclei and abundant dark brown granules (E: H&E staining).

Figure 7 Small animal PET imaging and pathologic examination of the pulmonary

metastasis mouse model bearing B16F10 melanoma cells. Two small lesions (< 2 mm) with high uptake of

18

F-PEG3-FPN in the lung were seen (A and C, red arrows).

Diffuse lung uptake was detected (B), with multiple lesions were seen in lung tissue samples (D). H&E (E, 200 ×) showed numerous neoplastic melanocytes with plentiful melanin deposits (black arrows).



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Figure 1 Reaction schemes to synthesize tosyl precursor and standard 19F-PEG3-FPN. Reagents and conditions: (i) Triethylene glycol, NaH, DMF, 100°C, 20 h; (ii) p-toluensulfonyl chloride, pyridine, room temprature, 4 h; (iii) TBAF, THF, 70°C, overnight; (iv) 18F-KF, K2,2,2 complex, DMSO, 100°C, 10 min. 92x57mm (600 x 600 DPI)


Page 26 of 32

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Figure 2 Identification and in vitro stability study of 18F-PEG3-FPN. (A) Analytic HPLC chromatograms after the co-injection of 18F-PEG3-FPN (red, retention time is 21.4 min) and standard unlabeled 19F-PEG3-FPN (blue, UV 254 nm). (B) 18F-PEG3-FPN stability in phosphate-buffered saline for 6 h. (C) and (D) 18F-PEG3FPN stability in human serum after incubation at 37°C for 3 and 6 h, respectively. 88x48mm (600 x 600 DPI)



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Figure 3 In vitro cell study. (A) In vitro studies of 18F-PEG3-FPN uptake by B16F10 melanoma cells and A375 amelanotic melanoma cells at 30 min, 60 min, 120 min. The differences in radioactivity over time in A375 cells are statistically insignificant (P > 0.1). (B) Blocking assay of 18F-PEG3-FPN by 19F-PEG3-FPN in B16F10 melanoma cells (P < 0.0001). 49x21mm (600 x 600 DPI)


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Figure 4 Dynamic PET imaging. (A) A series of coronal dynamic PET images obtained from mice bearing B16F10 melanoma tumors. (B) Time-activity curves of organs and tumors. 159x184mm (300 x 300 DPI)



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Figure 5 Static PET imaging of 18F-PEG3-FPN in primary subcutaneous melanoma model (1 h pi). Red arrows indicate tumors. Representative images of C57BL/6 mouse bearing B16F10 tumor xenograft (A), B16F10 tumor-bearing mouse blocked with unlabeled 19F-PEG3-FPN (B) and BALB/c mouse bearing A375 tumor xenograft. 109x55mm (300 x 300 DPI)


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Figure 6 Small animal PET imaging and tissue histologic changes of hepatic metastasis mouse model bearing B16F10 melanoma cells. (A) Representative coronal 18F-PEG3-FPN small animal PET images of liver metastatic lesions in liver (red arrows), with several black nodules < 0.5 cm were discovered in the liver tissue (C, red arrows). (B) Another mouse showed diffuse liver uptake on the PET imaging, with diffused lesions were seen in liver tissue samples (D). The pathology showed many cells with enlarged or multiple nuclei and abundant dark brown granules (E: H&E staining). 140x76mm (300 x 300 DPI)



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Figure 7 Small animal PET imaging and pathologic examination of the pulmonary metastasis mouse model bearing B16F10 melanoma cells. Two small lesions (< 2 mm) with high uptake of 18F-PEG3-FPN in the lung were seen (A and C, red arrows). Diffuse lung uptake was detected (B), with multiple lesions were seen in lung tissue samples (D). H&E (E, 200 ×) showed numerous neoplastic melanocytes with plentiful melanin deposits (black arrows). 140x76mm (300 x 300 DPI)


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Synthesis and Preclinical Evaluation of 18F-PEG3 ... - ACS Publications

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