A novel 99mTc-labelled glucose derivative for SPECT: A Promising

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A novel 99mTc-labelled glucose derivative for SPECT: A Promising Tumor Imaging Agent Xuran Zhang, Qing Ruan, Xiaojiang Duan, Qianqian Gan, Xiaoqing Song, Sian Fang, Xiao Lin, Jin Du, and Junbo Zhang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00415 • Publication Date (Web): 09 Jul 2018 Downloaded from http://pubs.acs.org on July 11, 2018

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Molecular Pharmaceutics

A novel 99mTc-labelled glucose derivative for SPECT: A Promising Tumor Imaging Agent Xuran Zhang†a,b,c, Qing Ruan†a,c, Xiaojiang Duana,c, Qianqian Gana,c, Xiaoqing Songa,c, Sian Fanga,c, Xiao Lina,c, Jin Dub, Junbo Zhanga,c* a: Key Laboratory of Radiopharmaceuticals (Beijing Normal University), Ministry of Education; College of Chemistry, Beijing Normal University, Beijing 100875, China b: Department of Isotopes, China Institute of Atomic Energy, P. O. Box 2108, Beijing 102413, China c: Beijing Shihong Pharmaceutical Center, Beijing Normal University, Beijing 100875, China

Abstract In this study, a D-glucosamine derivative with an isonitrile group (CN5DG) was synthesized and it was chosen to coordinate with 99m

99m

Tc for preparing

99m

Tc-CN5DG.

Tc-CN5DG could be readily obtained with high radiochemical purity (>95%) and

had great in vitro stability and metabolic stability in urine. The radiotracer demonstrated a positive response to the administration of glucose and insulin in S180 and A549 tumor cells in vitro, suggesting the mechanism of 99mTc-CN5DG into tumor cells was related to glucose transporters. Biodistribution studies in mice bearing A549 xenografts

showed

99m

Tc-CN5DG

had

a

high

tumor

uptake

and

high

tumor-to-background ratios. SPECT/CT images further supported its ability for tumor imaging. As a cheap, conveniently made and widely available probe, 99m

Tc-CN5DG would become a potential “working horse” and be a breakthrough

in 99mTc-labelled radiopharmaceuticals for tumor detection. Keywords: glucosamine-derivate ⋅ isonitrile ⋅ Tc-99m ⋅ SPECT ⋅ tumor imaging

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Introduction Cancer cells, being metabolic vigorous cells, show enhanced glucose metabolisms. Based on this, radiolabelled glucose derivatives have been applied for tumor imaging for many years.1-3 [18F]FDG (18F-2-fluoro-2-deoxy-D-glucose) is the most important radiolabelled glucose derivative for tumor diagnosis and it is regarded as the “molecule of the century” in the field of molecular imaging. However, in a lot of developing countries, the use of [18F]FDG is limited due to the need of a cyclotron for producing 18F isotope and high cost. By comparison, the numbers of single photon emission computed tomography (SPECT) scanners in the world are much more than those of positron emission tomography (PET) scanners,4 therefore, it is of great value to develop effective radiolabelled glucose analogs as SPECT tumor imaging agents. As a common radionuclide for SPECT,

99m

Tc has ideal physical and nuclide

properties (Eγ = 140 keV, T1/2 = 6.02 h), which are suitable for SPECT imaging and preparation of radiopharmaceutical. Moreover, technetium-99m has inexpensive cost and in-house availability. Up to date,

99m

Tc has become the most widely used

radionuclide for SPECT imaging. Recently, a number of

99m

Tc-labelled glucose

derivatives as potential tumor imaging agents have been developed.5-17 Among them, the

most

successful

complex

is

99m

Tc-ECDG

(ECDG:

ethylenedicysteine-deoxyglucose), which enters in Phase III clinical trials.6 However, the uptake of

99m

Tc-ECDG in tumor is low and its uptake in blood is high, thus

making its unsatisfactory tumor-to-blood ratio. Therefore, it is still worth developing novel 99mTc-labelled glucose derivatives with high tumor-to-background ratios. 2


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In general, native glucose bearing C-2 hydroxyl groups is not suitable ligand for complexing with

99m

Tc. To overcome the limitation, structural modification of

D-glucose or D-glucosamine becomes the feasible way to prepare novel 99m

Tc-labelled tumor imaging agents. Isonitrile (CN-R) is a monodentate ligand,

which can coordinate with ([99mTc(CN-R)6]+)

in

high

99m

Tc(I) core to form stable

yield.18-19

For

instance,

99m

99m

Tc complexes

Tc-MIBI

(MIBI:

2-methoxy-2-isobutyl isonitrile) is a monovalent cation with six same isonitrile ligands around 99mTc(I) core and exhibits good properties for myocardial imaging (Fig. 1).20 In this study, in order to develop novel

99m

Tc labelled glucose derivatives as

tumor imaging agents, a D-glucosamine derivative with an isonitrile group (CN5DG) was synthesized and labelled with 99m

99m

Tc to prepare

99m

Tc-CN5DG. The potential of

Tc-CN5DG for tumor imaging is evaluated for the first time.

Fig. 1. Chemical structures of MIBI, CN5DG, 99mTc-MIBI, 99mTc-CN5DG and Re-CN5DG 3



Experimental Section Materials and methods Chemical reagents and solvents were obtained from commercial sources. Na99mTcO4 was eluted from a commercial 99Mo/99mTc generator obtained from China Institute of Atomic Energy. Thin Layer Chromatography (TLC) was performed on a polyamide strip (Luqiao Sijia Biochemical Plastic Factory, Taizhou city, Zhejiang Province, China) and eluted with ammonium acetate (1 mol/L): methanol= 2:1(V/V). High Performance Liquid Chromatography (HPLC) was performed on a Waters 2489 system equipped with a Gabi raytest radioactivity detector. HPLC conditions: system 1 using an analytical column (Kromasil, 100-5 µm, 250 × 4.6 mm) at a flow rate of 1 mL/min (A, purified water with 0.1% trifluoroacetate; B, acetonitrile with 0.1% trifluoroacetate; 0-2 min, 10% B; 2-10 min, 10%-90% B; 10-18 min, 90%B; 18-25 min, 90%-10% B). System 2 using a semi-preparative column (Kromasil, 100-5 µm, 250 × 10 mm) at a flow rate of 4 mL/min (A, purified water with 0.1% trifluoroacetate; B, acetonitrile with 0.1% trifluoroacetate; 0-5 min, 10% B; 5-10 min, 10%-90% B; 10-15 min, 90%B; 15-20 min, 90%-10% B). NMR spectra were recorded on a 400 MHz Bruker Avance spectrophotometer. ESI-MS spectra were obtained on a LC-MS Shimadzu 2010 series. HRMS spectra were recorded on a Bruker microTOF-QII mass spectrometer and a Bruker solariX ESI mass spectrometer. Animals studies were performed in compliance with the guidelines of the Ethics Committee of Beijing Normal University and regulations on laboratory animals of Beijing Laboratory Animal Management Office. SPECT/CT studies were carried out 4


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on a micro SPECT/CT equipment (Trifoil, USA). Radioactivity was recorded on a Perkin-Elmer system (WIZARD2, 2480 Automatic Gamma Counter). Chemistry The synthesis route was shown in Fig. 2.

Fig. 2. Synthetic route of the CN5DG ligand. (a) formic acid, acetic anhydride, 95 oC; (b) 2,3,5,6-tetrafuorophenol, N, N'-dicyclohexylcarbodiimide, N, N-dimethylformamide, rt; (c) Burgess reagent, dichloromethane, 50 oC; (d) sodium hydroxide, D-glucosamine hydrochloride, CH3OH, rt.

Compounds 2, 3, 4 were synthesized according to the previous literature.21 The procedures were described below. 6-formamidohexanoic acid (2) 6-aminohexanoic acid (2 g, 19.06 mmol) was added into a 50 mL flask containing fomic acid (12 mL) and acetic anhydrate (6 mL). The flask was placed into an oil bath at 95 oC and the mixture was stirred for 4 h. The solvent was removed under vacuum and the residue was purified by a silica gel column chromatography using dichloromethane/methanol (5:1, V/V) as the mobile phase to give 2 as a slight yellow powder (1.68 g, 69%). IR (KBr)/cm-1：3329(NH)，1701, 1635 (C=O). 1H NMR (400 MHz, DMSO) δ 11.97 (s, 1H), 7.96 (m, 2H), 3.05 (dd, J = 12.9, 6.7 Hz, 2H), 2.18 (t, J 5



= 7.4 Hz, 2H), 1.55 – 1.44 (m, 2H), 1.39 (dt, J = 14.4, 7.1 Hz, 2H), 1.31 – 1.21 (m, 2H).

13

C NMR (100 MHz, DMSO) δ 174.42, 160.90, 36.94, 33.59, 28.75, 25.92,

24.17. ESI-MS, [M+H]+: m/z calcd. for C7H14NO3 160.09; found 160.15. 2,3,5,6-tetrafluorophenyl 6-formamidohexanoate (3) 2 (1.5 g, 9.42 mmol) and 2,3,5,6-tetrafluorophenol (1.88 g, 11.3 mmol) were mixed in N, N-dimethylformamide (DMF) (8 mL) and the mixture was reacted in an ice bath for 0.5 h. Then, N, N'-dicyclohexylcarbodiimide (DCC) (2.74 g, 14.13 mmol) was added to the mixture and stirred at room temperature overnight. The white precipitation in the reaction mixture was filtered and the filtrate was extracted by dichloromethane (50 mL) and water (50 mL) twice. The organic layer was collected and evaporated in vacuo to give a white residue. The residue was purified by a silica gel column chromatography using ethyl acetate/petroleum ether (2:1, V/V) as the mobile phase to afford 3 as a white solid (1.6 g, 55%). IR (KBr)/ cm-1: 3283 (NH), 1786, 1651 (C=O), 1084 (CF).

1

H NMR (400 MHz, CDCl3) δ 8.14 (s, 1H), 7.06 –

6.90 (m, 1H), 5.90 (s, 1H), 3.31 (q, J = 6.7 Hz, 2H), 2.67 (dd, J = 8.8, 5.7 Hz, 2H), 1.85 – 1.72 (m, 2H), 1.65 – 1.52 (m, 2H), 1.51 – 1.40 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 169.47, 161.43, 129.80-147.53 (m, Carom), 103.30 (t, J = 23 Hz, CaromH), 37.95, 33.31, 29.22, 26.12, 24.41. ESI-MS, [M+H]+: m/z calcd. for C13H14F4NO3 308.08; found, 308.11. 2,3,5,6-tetrafluorophenyl 6-isocyanohexanoate (4) 3 (1.2 g, 3.91 mmol) and Burgess reagent (1.40 g, 5.86 mmol) were mixed in CH2Cl2 (20 mL) and reacted at 50 oC for 4 h. The solvent was removed under vacuum 6


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and the oily residue was purified by a silica gel column chromatography using petroleum ether/ethyl acetate (10:1, V/V) as the mobile phase to afford 4 as a slight yellow oil (0.85 g, 75%). IR (KBr)/ cm-1: 2153(N≡C),1782(C=O), 1092(CF). 1H NMR (400 MHz, CDCl3) δ 6.99 (tt, J = 9.9, 7.1 Hz, 1H), 3.49 – 3.36 (m, 2H), 2.71 (t, J = 7.3 Hz, 2H), 1.88 – 1.68 (m, 4H), 1.66 – 1.50 (m, 2H).

13

C NMR (100 MHz,

CDCl3) δ 169.21, 156.45 (t, J = 5 Hz, NC), 129.75-147.40 (m, Carom), 103.33 (t, J = 23 Hz, CaromH), 41.41(t, J = 6 Hz, NCH2), 33.18, 28.82, 25.74, 23.99. ESI-MS, [M+H]+: m/z calcd. for C13H12F4NO2 290.07; found 290.10. 6-isocyano-N-(2,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)hex anamide (CN5DG, 5) D-glucosamine hydrochloride (496 mg, 2.31 mmol) and sodium hydroxide (2 mg, 2.31 mmol) were mixed in methanol (20 mL) and reacted at room temperature for 30 min. To this stirred mixture, 4 (1 g, 3.46 mmol) was added and the mixture continued to react at room temperature for 3 h. The solvent was evaporated under reduced pressure and the crude product was recrystallized by acetone to obtain the product 5 as a white powder (386 mg, 55%). IR (KBr)/ cm-1: 3295 (OH/NH), 2149 (N≡C), 1645 (C=O). 1H-NMR（400 MHz, D2O）: δ (ppm) 3.43-3.62 (m, 5H), 3.15-3.21 (m, 4H), 2.02-2.07 (m, 2H), 1.33-1.40 (m, 4H), 1.12-1.20 (m, 2H). 13C NMR (100 MHz, D2O) δ 177.07, 176.84, 149.86 (t, J = 7 Hz, NC), 94.60, 90.49, 75.53, 73.43, 71.14, 70.15, 69.75, 69.52, 60.34, 60.18, 56.09, 53.60, 41.01 (t, J = 6 Hz, NCH2), 35.40, 34.99, 27.50, 27.45, 24.57, 24.08. HRMS (ESI-TOF), [M+Na]+: m/z calcd. for C13H22N2O6Na 325.1370. found, 325.1374. 7



hexakis(((6-oxo-6-((2,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-y l)amino)hexyl)-l4-azanylidyne)methyl)rhenium(Ι) (Re-CN5DG) First, 50 mg of sodium citrate and 20 mg of L-cysteine were dissovled in 2 mL of water in a 10 mL penicillin vial, then 0.4 mL SnCl2⋅2H2O solution (100 mg SnCl2⋅2H2O /1mL 0.1 mol/L HCl) was added. The pH value of the above solution was adjusted to 5-6 with 0.1 mol/L NaOH and CN5DG (210 mg, 0.70 mmol) was then added to the mixture. Successively, KReO4 (10 mg, 0.035 mmol) was added. The resulting solution was heated at 100 °C for 60 min. After the reaction, the solution was analyzed by semi-preparative HPLC (system 2) and the desired compound (with a strong UV absorbance at 220 nm) that had nearly the same retention time with 99m

Tc-CN5DG was collected and concentrated to remove the solvent. The oily liquid

residue was dried and weighed (21 mg, 30%). IR (KBr)/ cm-1: 3314 (OH/NH), 2099 (N≡C), 1651 (C=O). 1H-NMR（400 MHz, D2O）: δ (ppm) 3.11-3.77 (m, 54H), 2.08-2.25 (m, 12H), 1.44-1.67 (m, 24H), 1.30-1.43 (m, 12H).13C NMR (100 MHz, D2O) δ 176.67, 176.40, 141.91, 94.82, 90.73, 75.71, 73.63, 71.33, 70.40, 69.99, 69.78, 60.55, 60.42, 56.35, 53.77, 48.66, 43.36, 35.57, 35.24, 28.63, 25.09, 25.03, 24.08. HRMS (ESI), [(C13H22N2O6)6ReH]2+: m/z calcd. for C78H133N12O36Re 1000.4246. found 1000.4264. Preparation of 99mTc-CN5DG Kit formulation for preparing 99mTc-CN5DG has been developed and applied in the biodistribution studies and SPECT imaging studies in mice bearing tumor. 1-3 mL of 99mTcO4- (37 MBq-1110 MBq) was added to a freeze-dried kit containing 1 mg of 8


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CN5DG, 0.06 mg of SnCl2⋅2H2O, 1 mg of sodium citrate and 1 mg of L-cysteine. The mixture (pH: 5~6) was incubated at 100 oC for 20 min. The radiochemical purity of 99m

Tc-CN5DG was determined by TLC or radio-HPLC (system 1) after cooling to

room temperature. Cell culture and tumor models Peking University Health Science Center (Beijing, China) provided the murine sarcoma S180 cell line and S180 cell line was cultured in Kunming mice ascites. A549 cell lines were obtained from Chinese Academy of Sciences Typical Culture Collection (Shanghai, China) and cultured in F-12K medium in a humidified atmosphere at 37 ℃ with 5% CO2. S180 tumor models were established by injecting S180 cells (2 × 106 in 0.1 mL saline) into the left upper limb armpit of Kunming mice (female, 18-22 g, 6 weeks). The tumors were allowed to grow 7-10 days with the diameter ranging from 5-8 mm. A549 tumor models were obtained by inoculating tumor cells (5 × 106 in 0.1 mL culture medium) subcutaneously into the right front flanks of Balb/C nude mice (female, 15-18 g, 5-6 weeks). As the tumor diameter was about 3-5 mm after 7-10 days’ inoculation, the mice bearing tumor can be used. Measurement of the octanol/water partition coefficient The partition coefficient of 99mTc-CN5DG was measured as the methods reported previously.16 Briefly, 50 µL (3.7 MBq) of the labelling solution was added to 750 µL of phosphate buffer solution (PBS, pH = 7.4, 0.025 mol/L) and the final solution was mixed with 800 µL 1-octanol. The mixture was vortexed for 2 min at room temperature and centrifuged at 2000 rpm for another 5 min. 100 µL (n = 3) from each 9



phase was collected and determined by γ-counter. The partition coefficient was calculated as follows: P = (radioactivity in octanol/radioactivity in aqueous layer). The partition coefficient (log P) was reported as average of three experiments. Stability studies in vitro and metabolic studies in vivo The stability of 99mTc-CN5DG in saline and mice serum in vitro were studied according to the methods reported previously.16 Mice serum was obtained from Kunming mice blood by centrifuging the blood at 10000 rpm for 3 min. 50 µL (3.7 MBq) of 99mTc-CN5DG was added in 200 µL mice serum or 200 µL saline and incubated at 37 oC. After 2 h in mice serum, 500 µL acetonitrile was added into the incubated solution. The precipitated protein was removed by centrifugation and the supernatant was passed through a 0.22 µm filter membrane. The filtrate was analyzed by HPLC with system 1. For stability in saline, the incubated solution was directly analyzed by HPLC after incubation for 6 h. For the metabolic stability study in urine, Kunming normal mice were injected 18 MBq of 99mTc-CN5DG. At 30 and 60 min after injection, urine was collected and centrifuged at 3000 rpm for 5 min. The supernatant was analyzed by HPLC with system 1. Cell uptake studies Cell uptake studies were carried out on S180 and A549 cells. S180 cells were suspended in glucose-free DMEM culture medium (6 × 106 cells/mL) and added to a 24-well plate (0.5 mL per well). For A549 cells, the cells were seeded in a 24-well plate (3 × 105/each well) 24 h before the experiment. To each well (n = 6), 480 kBq of 99m

Tc-CN5DG in 500 µL of glucose-free DMEM culture medium was added and the 10


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mixture was incubated at 37 oC for 4 h. To evaluate if the uptake of radiotracer was related to glucose transporters, glucose (D-glucose or L-glucose, 2 mg) or insulin (2 units) and 480 kBq of 99mTc-CN5DG were added to wells (n = 6) containing S180 cells or A549 cells and incubated for 4 h. After incubation, S180 cells were collected in 2 mL tubes and the tubes were centrifuged at 8000 rpm for 5 min and washed with 1 mL of cold PBS (0.025 mol/L, pH = 7.4) twice. A549 cells were first washed (2 × 1 mL) with cold PBS (0.025 mol/L, pH = 7.4) and then lysed with 1 mL of 1 mol/L NaOH. The radioactivity of the cells and the total added radioactivity were counted by a Perkin Elmer γ-counter. The percentage of cell uptake can be expressed as cells counts /total added counts. Biodistribution studies Biodistribution studies were performed in A549 tumor-bearing mice. Mice were injected 99mTc-CN5DG (74 kBq, 0.1 mL) via tail vein and sacrificed at 30 min, 60 min and 120 min after injection. The interested organs, blood and tumor were removed, weighted, and the radioactivity was measured. The values are expressed in terms of the percentage of injected dose per gram (%ID/g) of tissue. By comparison, mice were injected

18

F-FDG (0.37 MBq, 0.1 mL) via

tail vein and sacrificed at 60 min after injection. The interested organs, blood and tumor were removed, weighted, and the radioactivity was measured. In order to determine that the uptake of 99mTc-CN5DG was related to a glucose-mediated process, S180 tumor-bearing mice were injected intravenously with saline (0.1 mL, n = 4) or 2-deoxy-D-glucose (6 mg per mouse, n = 4) or 11



intramuscularly with insulin (0.25 unit per mouse, n = 4) 30 min before the injection of 99mTc-CN5DG (74 kBq, 0.1 mL). These mice were sacrificed at 60 min after the injection of 99mTc-CN5DG. Small animal SPECT/CT imaging Micro SPECT/CT imaging scans were taken in nude mice bearing A549 xenografts with different tumor size (3 mm in one group, n = 3; 5 mm in another group, n = 3). Each mouse was injected

99m

Tc-CN5DG (55 MBq, 0.1 mL)) via tail

vein. The SPECT/CT imaging scans were acquired at 30 min, 60 min and 120 min after injection. The mice were imaged following a SPECT/CT protocol after anesthetized with inhalation of 1.5% isoflurane. The CT scan (512 views, 2×2 binding, 75 kV, exposure time 230 ms) was first carried for 4 min followed by a SPECT acquisition (Peak 140 keV, 20% width, 90 degrees rotation, MMP 919 collimator) for 15 min. The SPECT/CT images were acquired by using the HiSPECT software and the vivoquant 2.5 software. Statistical analysis Quantitative data are presented as the average ± SD (standard deviation). Statistical analysis was performed with EXCEL 2010 software using the t-test. The statistical tests were two-tailed. P < 0.05 represented statistically significant.

Results Chemistry The synthesis of the glucose derivative (CN5DG) containing an isonitrile was shown in Fig. 2. First, 6-aminohexanoic acid was N-formylated to obtain 2, and then the 12


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carboxyl of 2 was converted to active ester 3 by reacting with 2,3,5,6-tetrafluorophenol (TFP). The active ester 3 was dehydrated using burgess reagent to obtain 4 as an effective bifunctional coupling agent.22 In the final step, the isocyanide-containing active ester 4 was conjugated with D-glucosamine hydrochloride under basic condition. In order to obtain pure product, the amount of 4 was 1.5 times that of D-glucosamine hydrochloride to make sure that all the D-glucosamine hydrochloride was reacted. The amide linker in CN5DG molecule was chosen because of its good stability in biological conditions. The structure of CN5DG was confirmed by IR, 1HNMR, 13C-NMR and HRMS. Radiochemistry The radiochemical purity of

99m

Tc-CN5DG was over 95% analyzed by HPLC as

shown in Fig. 3 (red color). The retention time of

99m

Tc-CN5DG and

99m

TcO4- was

10.18 and 3.93 min (99mTcO4- not given), respectively. Because Tc and Re exhibit group homology, in the typical practice for preparing

99m

Tc radiopharmaceutical, the

corresponding rhenium complex is often prepared to confirm the structure of the 99m

Tc-labelled complex. Re-CN5DG was synthesized for the structure confirmation of

99m

Tc-CN5DG (Fig. 1). The structure of Re-CN5DG was confirmed by IR,

1

HNMR,

13

C-NMR and HRMS. After co-injection 99mTc-CN5DG and Re-CN5DG,

the HPLC pattern (system 1) of Re-CN5DG (9.96 min) nearly matched with the corresponding

99m

Tc-CN5DG (10.18 min) (Fig. 3), suggesting

99m

Tc-CN5DG

possessed the proposed structure. In order to determine the radiochemical purity quickly, TLC can be routinely used. The Rf value of 99mTc-CN5DG was 0.7-1.0 while 13



that of

Page 14 of 27

99m

TcO4- and 99mTcO2 ⋅nH2O was 0-0.1. The specific activity was calculated to

be 11.17-335.22 GBq/mmol. The log P of 99mTc-CN5DG was -3.57 ± 0.35, suggesting that the radiotracer was hydrophilic. Stability studies presented in Fig. 4 showed that the radiotracer was stable in saline at room temperature for 4 h and in mice serum at 37 oC for 2 h and had no other metabolite in mice urine in vivo, suggesting 99m

Tc-CN5DG exhibited good stability.

Fig. 3. Co-injection analysis of Re-CN5DG (black color) and

99m

Tc-CN5DG (red color)

using analytical RP-HPLC (system 1)

Fig. 4. HPLC profiles (system 1) of

99m

Tc-CN5DG in (A) saline for 4 h and (B) mice

serum for 2 h in vitro. Urine metabolic studies at (C) 30 min and (D) 60 min in vivo 14


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In vitro cellular uptake study The uptake data of

99m

Tc-CN5DG in S180 and A549 tumor cells were

shown in Fig. 5. The blocking studies with 2 mg of D-glucose suggested that the uptake of

99m

Tc-CN5DG on both cells could be significantly blocked (for

S180 cells, 41%, P = 0.0003; for A549 cells, 47%; P = 0.002), while 2 mg of L-glucose had no significant influence on the uptake (for S180 cells, P = 0.567; for A549 cells, P = 0.189). When 2 units of insulin was administered, the uptakes on both cells increased significantly (for S180 cells, 69%, P = 0.005; for A549 cells, 70%, P = 0.02). These findings demonstrated that 99m

Tc-CN5DG was transported via the glucose transporters.

Fig. 5. Cell uptake data of 99mTc-CN5DG in (A) S180 and (B) A549 tumor cells when glucose (L-glucose or D-glucose, 2 mg) or insulin (2 units) was co-incubated (*P < 0.05)

Biodistribution studies The biodistribution results of biodistribution of

99m

Tc-CN5DG and

99m

Tc-CN5DG and comparisons of

18

F-FDG in nude mice bearing A549

xenografts at 1 h after injection were shown in Fig. 6. As demonstrated in Fig. 6A, the tumor had a high uptake from 30 min after injection to 120 min after 15



Page 16 of 27

injection. The tumor-to-background ratios retained high levels at all the time studied and increased with time. The high accumulation of

99m

Tc-CN5DG in

kidney suggested that the tracer was cleared via the urinary tract. When compared to than

18

F-FDG (Fig. 6B), as for the tumor uptake,

18

F-FDG is higher

99m

Tc-CN5DG. However, the tumor-to-blood, tumor-to-muscle and

tumor-to-lung ratios of

99m

Tc-CN5DG are superior to those of

18

F-FDG at 1 h

post-injection (Fig. 6C).

Fig. 6. (A) Biodistribution data of

99m

Tc-CN5DG in nude mice bearing A549 xenografts (n

= 3) at 30 min, 60 min and 120 min after injection. (B) Comparision of biodistribution of 99m

Tc-CN5DG and

18

F-FDG in nude mice bearing A549 xenografts at 60 min after

injection (n = 3). (C) Comparision of the tumor/muscle, tumor/blood and tumor/lung ratios 16


Page 17 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


of 99mTc-CN5DG and 18F-FDG. (D)Tumor and blood uptake data of 99mTc-CN5DG in mice bearing S180 tumor at 1 h after injection when saline (control), 2-deoxy-D-glucose (2 mg) or insulin (0.25 unit) was pretreated (%ID/g ± SD, n = 4). Significant changes can be found as compared with the control group (*P < 0.05)

As shown in Fig. 6D, pretreatment with 2-deoxy-D-glucose could block the tumor uptake (32%, P = 0.022) of

99m

Tc-CN5DG in S180 tumor-bearing mice,

and insulin could enhance tumor uptake (111%, P = 0.015), suggesting 99m

Tc-CN5DG exhibited the characters of glucose-mediated process into

tumors.

Small animal SPECT/CT imaging The small animal SPECT/CT imaging using

99m

Tc-CN5DG was performed

in A549 xenografts-bearing mice as shown in Fig. 7. From the whole body SPECT/CT images, A549 tumors could be clearly visualized from 30 min to 120 min post-injection. These SPECT/CT images corresponded with the biodistribution results in mice. Kidney and bladder could also be visible due to the high hydrophilicity of

99m

Tc-CN5DG. Radioactivity uptake was low in the

thyroid and stomach, suggesting in vivo stability of 99mTc-CN5DG.

17



Fig. 7. Whole body SPECT/CT images of

99m

Tc-CN5DG (55 MBq) in nude mice bearing

A549 xenografts at 30 min, 60 min and 120 min after injection. (A) Nude mice bearing A549 xenografts (n = 3) were imaged with a tumor size of 3 mm in diameter. (B) Nude mice bearing A549 xenografts (n = 3) were imaged with a tumor size of 5 mm in diameter. (C) The A549 tumor used in (A) was seperated from the mouse and measured by a Vernier caliper.

Discussion 99m

Tc, as a generator produced radionuclide, is readily available and affordable.

Moreover, the 186/188Re analogs of 99mTc-labelled tracer can afford therapeutic tactics. Thus, to develop novel

99m

Tc-labelled glucose derivatives as tumor imaging agents

still has great significance. To discover insertion of

99m

99m

Tc-lablled glucose derivatives, the biggest problem is the

Tc-bearing groups to a glucose molecule perhaps changes the

biochemical properties of glucose. As glucose and glucosamine are difficult to chelate 18


Page 18 of 27

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with 99mTc to form stable 99mTc complexes, feasible strategy to solve the problem is to design glucose derivatives having a suitable bifunctional chelator. The choice of a suitable bifunctional chelator which can link to the glucose molecule and coordinate with

99m

Tc is important for preparing desired

99m

Tc-labelled glucose derivatives as

tumor imaging agents.12 It is should be noted that both 18F-FDG and 99mTc-ECDG are radiolabelled glucose derivatives modified at the C-2 position of the D-glucose. Previous studies also suggested the design of a novel ligand should be restricted to glucose analogs modified at the C-2 position, as this was regarded as the most suitable site to tolerate structure alteration.18 Isonitrile (CN-R) is able to coordinate with 99m

Tc(I) core to prepare [99mTc(CN-R)6]+ in high yield. Considering the above factors,

we skillfully designed and synthesized a novel glucosamine derivative (CN5DG), containing

an

isonitrile

through

the

reaction

of

D-glucosamine

with

isocyanide-containing active ester. The preparation of

99m

Tc-CN5DG by using a freeze-dried kit formulation is easy

and convenient. The RCP of the product was over 95% and there was no need for further purification. The structure of 99m

99m

Tc-CN5DG would be similar to that of

Tc-MIBI, having six CN5DG ligands around technetium-99m (+1) to form a

regular octahedron structure.20 The proposed structure of

99m

Tc-CN5DG was

successfully verified by the corresponding Re-CN5DG complex. 18

F-FDG enters cells mainly by GLUT1 and its uptake could be inhibited

by D-glucose and enhanced by insulin.23-24 In order to investigate the uptake mechanism of

99m

Tc-CN5DG, we carried out cellular uptake studies. In our 19



Page 20 of 27

experiments in both cell lines, D-glucose can significantly inhibit the uptake of 99m

Tc-CN5DG and L-glucose had no effect on

99m

Tc-CN5DG uptake.

Moreover, insulin can increase the uptake of 99mTc-CN5DG. The primary cell uptake studies suggested that the uptake mechanism of 18

99m

Tc-CN5DG and

F-FDG was similar. This view was supported by our biodistribution studies in

mice bearing S180 tumor. The mice bearing S180 tumor pretreated intravenously with 2-deoxy-D-glucose exhibited decreased tumor uptake of 99m

Tc-CN5DG, while pretreated intramuscularly with insulin increased tumor

uptake of

99m

Tc-CN5DG. Insulin, as a blood sugar level regulator, could

decrease the sugar concentration in blood. As demonstrated in Fig. 6D, the uptake of

99m

Tc-CN5DG in blood was significantly decreased (53%, P =

0.044). These findings further proved that GLUT1. Biodistribution studies of

99m

99m

Tc-CN5DG was transported via

Tc-CN5DG in mice bearing A549

xenografts at 30 min, 60 min and 120 min after injection demonstrated that the tracer had a rapid, high tumor uptake and cleared quickly from normal organs. At 30 min after injection, all organs or tissues reached the maximum uptake and decreased as a function of time. Except for the obvious uptakes of kidney and tumor, the uptakes of other organs are low, thus making it feasible to obtain satisfactory target/non-target ratios. When compared to uptake of

99m

Tc-CN5DG is a slight lower than that of

18

18

F-FDG, the tumor

F-FDG. However, due

to the much higher uptakes of lung, muscle and blood of

18

F-FDG, the

tumor/blood (19.83 ± 4.39), tumor/muscle (14.37 ± 6.96) and tumor/lung (5.22 20


Page 21 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


± 0.58) ratios of

99m

Tc-CN5DG are far better than those of

18

F-FDG (8.40 ±

3.89, 0.32 ± 0.08 and 1.19 ± 0.29) at 1 h post-injection. Yang et al. also described the biodistribution results of [18F]FDG in mice bearing A549 xenografts,5 thus making it possible for us to compare the biodistribution data of

99m

Tc-CN5DG and [18F]FDG in mice bearing the same xenografts. When

compared to

18

F-FDG, in terms of the tumor uptake, there are no significant

differences between

99m

Tc-CN5DG and

18

F-FDG (2.35 ± 0.27 %ID/g vs 2.23 ±

0.15 %ID/g at 30 min after injection and 1.48 ± 0.23 %ID/g vs 1.70 ± 0.17 %ID/g at 120 min after injection). With regard to tumor-to-muscle, tumor-to-blood and tumor-to-lung ratios, 18

99m

Tc-CN5DG are far better than

F-FDG (10.22, 5.11 and 2.80 vs 0.46, 2.82 and 0.91 at 30 min post-injection;

24.67, 49.33 and 13.45 vs 0.32, 7.26 and 0.78 at 120 min post-injection). Considering the structure similarity between

99m

Tc-CN5DG and

99m

Tc-MIBI, we

conducted the biodistribution studies in mice bearing S180 tumor of the two complexes (n = 5). The tumor uptake of 99mTc-CN5DG was 1.07 ± 0.21 %ID/g, while that of 99mTc-MIBI was 0.83 ± 0.33 %ID/g. The uptakes of 99mTc-MIBI in heart, liver, kidney and muscle were 9.38 ± 2.21 %ID/g, 7.66 ± 2.04 %ID/g, 14.63 ± 1.32 %ID/g and 3.89 ± 1.10 %ID/g, while those of 99mTc-CN5DG were 0.41 ± 0.02 %ID/g, 0.38 ± 0.08 %ID/g, 2.68 ± 0.32 %ID/g and 0.27 ± 0.10 %ID/g, respectively. These findings showed that the two tracers had different metabolic routes in vivo. Glucose groups in 99m

Tc-CN5DG make it hydrophilic, whereas six alkyl groups in

99m

Tc-MIBI cause it

lipophilic. We speculate that lipophilicity plays an important role in influencing the 21



biodistribution data of the complexes. As a tumor imaging agent, superior to

99m

Page 22 of 27

99m

Tc-CN5DG is

Tc-MIBI because the former has lower uptakes in heart, liver, kidney

and muscle. SPECT/CT imaging studies in A549 xenografts-bearing mice showed 99m

Tc-CN5DG can clearly visualize the tumor sites from 30 min to 120 min

after its administration. The tumor volume that can be detected by using 99m

Tc-CN5DG was about 3 mm, suggesting

99m

Tc-CN5DG holds the potential

to be used in molecular imaging to early diagnose tumor and monitor treatment efficiency.

Conclusion In summary, CN5DG was skillfully designed and synthesized.

99m

Tc-CN5DG

could be easily prepared using a CN5DG kit in high radiolabelling yield (>95%). Uptake mechanism studies in vitro and in vivo suggested that it was transported by GLUTs to enter the tumor cells. Preclinical studies demonstrated

99m

Tc-CN5DG

would be a powerful tool for tumor detection, justifying further investigation. It is reasonable to conclude that 99mTc-CN5DG will make a positive impact on oncology imaging.

AUTHOR INFORMATION Corresponding Author *[email protected] ORCID: Junbo Zhang: 0000-0003-3549-6483 Present Addresses 22


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No. 19 Xinjiekou Wai Boulevard, Haidian District, Beijing. 100875, P. R. (China). Author Contributions †Xuran Zhang and Qing Ruan contribute equally. Notes The authors declare no conflicts of interest. ACKNOWLEDGEMENTS The work was supported by the National Natural Science Foundation of China (21771023) and the project of Beijing Municipal Science and Technology Commission (Z181100002218033). The authors give thanks to Prof. Zhanbin Zhang for his suggestions on the organic synthesis of the compounds. REFERENCES (1) Hicks, R.J. Role of 18F-FDG PET in assessment of response in non-small cell lung cancer. J. Nucl. Med. 2009, 50, 31S-42S. (2) Jaini, S.; Dadachova, E. FDG for therapy of metabolically active tumors. Semin. Nucl. Med. 2012, 42, 185-189. (3) Liu, T.L.; Zhang, J.B.; Wang, X.B.; et al. Radiolabeled glucose derivatives for tumor imaging using SPECT and PET. Curr. Med. Chem. 2014, 21, 24-34. (4) Paez, D.; Orellana, P.; Gutierrez, C.; et al. Current status of nuclear medicine practice in Latin America and the Caribbean. J. Nucl. Med. 2015, 56, 1629-1634. (5) Yang, D.J.; Kim, C.G.; Schechter, N.R.; et al. Imaging with 99mTc ECDG targeted at the multifunctional glucose transport system feasibility study with rodents. Radiology 2003, 226, 465-473. 23



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Table of Content graphic

99m

Tc-CN5DG is a promising tumor imaging agent being cheaper and readily available for routine

use and would be a breakthrough in 99mTc radiopharmaceuticals.

27


A novel 99mTc-labelled glucose derivative for SPECT: A Promising

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