Applied Radiation and Isotopes 77 (2013) 166–173

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Applied Radiation and Isotopes

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E-mai(C.-L. Ch

journal homepage: www.elsevier.com/locate/apradiso

Evaluation of 131/123I-5-iodo-2′-deoxycytidine as a novel proliferationprobe in a tumor mouse model

Wen-Yi Chang a, Wei-Ti Kuo a, Chun-Yi Wu a, Chih-Yuan Lin a,d, Pei-Chia Chan a,Chih-Chieh Shen e, Ren-Shan Liu a,b,c, Hsin-Ell Wang a,n, Chuan-Lin Chen a,nn

a Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, Taipei, Taiwanb Department of Nuclear Medicine, Faculty of Medicine, National Yang-Ming University, Taipei, Taiwanc Department of Nuclear Medicine and National PET/Cyclotron Center, Taipei Veterans General Hospital, Taipei, Taiwand Institute of Nuclear Energy Research, Department of isotope application, Taoyuan, Taiwane Section of Nuclear Medicine, Cheng Hsin General Hospital, Taipei, Taiwan

H I G H L I G H T S

� N.c.a. [123I]ICdR and [123I]IUdR can be prepared from their organotin precursors.

� Both SPECT imaging and biodistribution show specific tumor uptake of [123I]ICdR.� [123I]ICdR is a plausible SPECT probe for imaging tumor proliferation.� Metabolic instability of [123I]IUdR in vivo limits its clinical application.

a r t i c l e i n f o

Article history:Received 6 June 2012Received in revised form1 February 2013Accepted 10 March 2013Available online 25 March 2013

Keywords:5-Iodo-2′-deoxycytidine5-Iodo-2′-deoxyuridineProliferationSPECT

43/$ - see front matter & 2013 Elsevier Ltd. Ax.doi.org/10.1016/j.apradiso.2013.03.021

espondence to: Department of Biomedicas, National Yang-Ming University, No. 155, STaipei 112, Taiwan. Tel.: þ886 2 28267215; farespondence to: Department of Biomedicas, National Yang-Ming University, 155, Sec. 2

l addresses: hewang@ym.edu.tw (H.-E. Wang)en).

a b s t r a c t

This study evaluated a radioiodinated deoxycytidine analog, 131I-5-iodo-2′-deoxycytidine ([131I]ICdR), asa novel proliferation probe and compared it with 131I-5-iodo-2′-deoxyuridine ([131I]IUdR) in a NG4TL4sarcoma-bearing mouse model. As an imaging agent, the biological characteristics of [123I]IUdR is notsatisfactory due to its metabolic instability and short biological half-life in vivo. With [123I]ICdR/SPECT itwas possible to clearly delineate the tumor lesion at 1 h post-injection (tumor-to-muscle ratio 7.74) intumor-bearing mice. The results of biodistribution were consistent with those observed in scintigraphicimaging. This study demonstrated that [131I]ICdR is a more promising SPECT probe than [131I]IUdR forimaging proliferation.

& 2013 Elsevier Ltd. All rights reserved.

1. Introduction

The rate of malignant cell division is usually uncontrolled, and is animportant characteristic that can be addressed for tumor detection.Positron emission tomography (PET) and single photon emissioncomputed tomography (SPECT) are imaging techniques frequentlyused in oncology for detecting and staging viable and proliferatingtumor. PET or SPECT with a probe that is specific for cell proliferationwould greatly help locate the tumors and/or assess the therapeuticefficacy of treatment (Josephs et al., 2009; Saga et al., 2009).

ll rights reserved.

l Imaging and Radiologicalec. 2, Linong Street, Beitoux: þ886 2 28201095.l Imaging and Radiological, Linong Street, Taipei 11217,

, clchen2@ym.edu.tw

18F-Fluorodeoxyglucose (18F-FDG), the most widely used noninva-sive tracer, is neither specific nor selective for tumor proliferationdetection. For instance, macrophages, which invade tumor and alsofound in inflammatory lesion, may exhibit its increased uptake(Bakheet et al., 2000; van Waarde et al., 2004) as well as tumors.

Recently, monitoring DNA synthesis is considered a more specificapproach to image tumor because nucleosides are the essentialsubstrates for cell proliferation (Deng et al., 2004). Cells always needdeoxythymidine monophosphate (dTMP) to support the synthesis ofDNA sequence either by methylating deoxyuridine (“de novo” path-way) or by phosphorylating thymidine imported from extracellularmilieu (“salvage” pathway) (Schwartz et al., 2003).

3′-Deoxy-3′-18F-fluorothymidine ([18F]FLT), a [18F]–labeled thymi-dine analog, has been developed as a PET tracer to image prolifera-tion in vivo and was considered the most suitable proliferation probefor PET (Shields et al., 1998). [18F]FLT is retained in proliferatingtissues through the activity of thymidine kinase, an enzymeexpressed during the DNA synthesis phase of the cell cycle (Raseyet al., 2002). However, F-18 atom of [18F]FLT is a substituent for the

W.-Y. Chang et al. / Applied Radiation and Isotopes 77 (2013) 166–173 167

hydroxyl group of thymidine, the site which polymerase binds to, so[18F]FLT is not a direct marker for DNA synthesis because most FLTcannot be incorporated into DNA. Therefore, [18F]FLT measures onlycellular thymidine kinase activity, although phosphorylation andthus trapping of FLT correlate with proliferation (Vesselle et al.,2002). For instance, if TK1 remains high in S-phase cells regardless ofwhether these cells are proliferating, then high tracer uptake levelmight not always track with rapid proliferation.

Several radiolabeled nucleoside analogs, e.g. [18F]FLT (Badingand Shields, 2008; Menda et al., 2009; Murayama et al., 2009;Rasey et al., 2002; Vesselle et al., 2002), [18F]FMAU (Lu et al.,2002; Sun et al., 2005a, 2005b) and 18F-FUdR (Kameyama et al.,1995; Wang et al., 2006), have been investigated as tumordetection probes. FMAU (2′-deoxy-2′-fluoro-5-methyl-β-D-ara-bino-furanosyluracil) can be labeled with either C-11 or F-18and was considered a promising proliferation tracer. Previousstudies have shown that FMAU is incorporated into DNA but notinto RNA or protein and resists degradation. The primarylimitation of [11C/18F]FMAU appears to be that it is a relativelygood substrate for TK2 and may exhibit appreciable accumula-tion in mitochondrion-rich human myocardium. The higherbackground incorporation into mitochondrial DNA may render[11C/18F]FMAU a less sensitive probe than [18F]FLT in monitoringcell proliferation with PET.

5-Fluoro-2′-deoxyuridine (FUdR), another thymidine analogwhich can be phosphorylated by the host uridine kinase toFUdR-monophosphate (FUdR-MP). FUdR-MP is entrapped withinthe target cell, acts as an irreversible inhibitor of thymidylatesynthase (a key enzyme in de novo pathway) and results inincreased DNA synthesis via salvage pathway (Abe et al., 1983).A common method for [18F]FUdR preparation is via electrophilicfluorination of its TUdR (1-(2-deoxy-β-D-ribofuranosyl)-5-tributyl-stannyluracil) precursor and gives low specific activity [18F]FUdRproduct due to the inevitable addition of cold F2 in producing [18F]F2 as fluorinating agent (Chang et al., 2009). The co-existence of asignificant amount of cold FUdR in [18F]FUdR injection willinfluence the route of DNA synthesis in target cells and tissues,and thus the observed [18F]FUdR accumulation may not representthe actual proliferation status of target tissues. Another majorlimitation of [18F]FUdR in tumor monitoring is its rapid defluor-ination in vivo (Bading and Shields, 2008).

The development of proliferation-specific probes for SPECT ima-ging has received much less attention. SPECT probes have someadvantages compared with those used with PET, including lower costfor radiopharmaceutical, wide-spread availability of imaging facilities,easier radiotracer synthesis, and longer physical half-life of SPECTradionuclides that facilitate their clinical use for determining the slowbiological activities in living bodies (Meikle et al., 2005).

Iododeoxyuridine (IUdR) is a thymidine (TdR) analog in whichthe 5-methyl group of TdR is replaced by iodine. IUdR resemblesTdR in biological behavior due to similar van der Waal radi ofmethyl moiety and iodine atom. IUdR is incorporated into thenuclear DNA of dividing cells exposed to the agent during the Sphase of the cell cycle (Neshasteh-Riz et al., 1998). Intravenousadministration of radioiodinated iododeoxyuridine (nIUdR), how-ever, is unlikely to be useful as a tumor imaging agent becauseof the non-specific uptake by all proliferating cells and rapiddehalogenation in vivo (T1/2o5 min in human,o7 min in mice)(Kasis AI et al., 1996; Mariani G et al., 1996). Previous studiesin experimental animals and human subjects have shown thatefficient tumor targeting can be obtained after locoregionaladministration (Kasis AI et al., 1996; Mariani G et al., 1996). Severalclinical studies in patients with liver metastases from colorectalcancer have demonstrated significant tumor uptake after nIUdRinfusion through the hepatic artery (Daghighian et al., 1996;Macapinlac et al., 1996). In addition, Roelcke et al. also showed

that 124I-IUdR is feasible for detecting malignant glioma with PETimaging (Roelcke et al., 2002).

Because of the short biological half-life of [123/131I]IUdR, thereare practical difficulties in achieving satisfactory tumor targetingafter systemic administration in patients for certain tumor types(Kassis, 1990). Iododeoxycytidine (ICdR), a cytidine analog, is alsoable to incorporate into DNA sequence. The amount of ICdRincorporated into DNA was similar to that of IUdR in mouse, butwas more slowly degraded than IUdR in rat (Kriss et al., 1963,1962). The study described herein has demonstrated that [123I]ICdR has the potential as a SPECT probe for clinical tumor imaging.

2. Experimental

2.1. Reagents and instrumentation

2′-Deoxycytidine hydrochloride, bis(tributyltin), hydrogen perox-ide, dimethylformamide (DMF), acetonitrile (ACN) and ethanol werepurchased from Sigma-Aldrich Chemical (St. Louis, MO, USA). Iodineand methanol were purchased from Merck and Co., Inc. Silvertrifluoroacetate, tris(dibenzylideneacetone) dipalladium(0)((dba)3Pd2), 5-iodo-2′-deoxyuridine (IUdR), Celite® 545, sodiumthiosulfate and sodium bicarbonate were purchased from ACROSOrganics (Morris Plains, NJ, USA). Acetic acid and HCl were purchasedfrom J. T. Baker Inc (Phillipsburg, NJ, USA). All solvents were driedbefore use by distillation from sodium or calcium hydride. The NMRspectra were recorded with Bruker 400 UltraShield at a protonfrequency of 400 MHz and chemical shifts were expressed in ppm.An imaging scanner (AR-2000; Bioscan, Washington, D.C., USA) wasused to scan thin-layer chromatography (TLC) plates and to analyzethe chromatograms. High resolution mass spectra were measured ona FINNIGAN MAT 95S Mass Spectrometer (Germany). High perfor-mance liquid chromatography (HPLC) was performed with a Waters600 controller (Milford, MA, USA) equipped with aWaters TM486 UVdetector (wavelength 254 nm) (Milford, MA, USA) and a flowthrough radioactivity detector (Flow Count FC-004; Bioscan,Washington, D.C., USA). The radioactivity was measured with aCRC-15 R dose calibrator (CAPINTEC Inc, USA). Minimum essentialmedium (MEM), fetal bovine serum (FBS) were purchased from LifeTechnologies, Inc (Carlsbad, CA, USA).

2.2. 5-Iodo-2′-deoxycytidine (ICdR, 2) (Scheme 1)

Iodine (670 mg; 2.6 mmol) and silver trifluoroacetate (583 mg;2.6 mmol) were added to a solution of 2′-deoxycytidine hydro-chloride and the mixture was stirred at 35 1C for 20 h. The mixturewas passed through celite and the solvent removed using a rotaryevaporator. The crude product was purified by silica gel chroma-tography (eluent: CH2Cl2/methanol¼20/1 to 4/1) to afford ICdR (2)as a white crystalline solid (370 mg, yield 60%).

1H-NMR (MeOH-d4, 400 MHz): δ 8.59 (s, 1H, H-6), 6.17(t, J¼6.0 Hz, 1H, H-1′), 4.37 (m, 1H, H-3′), 3.94 (m, 1H, H-4′),3.78(m, 2H, H-5′), 2.27 (m, 2H, H-2′); 13C NMR (MeOH-d6,100 MHz): δ 164.9 (C, CQO), 155.8 (C, C-4), 152.1 (CH, C-6),149.9 (C, C-5), 89.1 (CH, C-1′), 88.0 (CH, C-3′), 71.4 (CH, C-4′),62.1 (CH2, C-5′), 42.3 (CH2, C-2′); Exact mass (HRMS) calcd forC9H12IN3NaO4

þ , 375.9770; found 375.9780

2.3. 1-(2-Deoxy-β-D-ribofuranosyl)-5-tributylstannylcytosine(TCdR, 3)

A mixture of ICdR (353 mg, 1.0 mmol), (dba)3Pd2 (30.86 mg,0.03 mmol) and bis(tributyltin) (1.44 g, 2.49 mmol, 1.26 mL) inDMF (4 mL, 0.25 M) was heated at 65 1C for 5 h. The reactionmixture was filtered through celite and the solvent was removed

Scheme 1. Synthesis of authentic ICdR (2) and the precursor TCdR (3) for conducting radioiodination .

Scheme 2. Synthesis of [123/131I]ICdR and [123/131I]IUdR.

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by rotary evaporator under reduced pressure. The crude oilyproduct was purified by silica gel chromatography (eluent: chloro-form/methanol¼10/1) to afford 206.5 mg of 3 (40%).

1H-NMR (CDCl3, 400 MHz): δ 7.85 (s, 1H, H-6), 6.3 (t, J¼6.8 Hz,1H, H-1′), 4.37 (m, 1H, H-3′), 3.96 (m, 1H, H-4′), 3.73(m, 2H, H-5′),2.39~2.08 (m, 2H, H-2′), 1.63∼0.88 (m, 27 H, SnBu3); 13C NMR(CDCl3, 100 MHz): δ 171.1 (C, C¼O), 158.3 (C, C-4), 148.8 (CH, C-6),102.9 (C, C-5), 89.2 (CH, C-3′), 87.7 (CH, C-1′), 72.5 (CH, C-4′), 62.9(CH2, C-5′), 42.2 (CH2, C-2′), 30.0 (CH2, SnBu3), 28.2 (CH2, SnBu3),14 (CH3, SnBu3), 10.5 (CH2, SnBu3); Exact mass (HRMS) calcd forC21H40N3O4Snþ , 518.2041; found 518.2079

2.4. 1-(2-Deoxy-β-D-ribofuranosyl)-5-tributylstannyluracil (TUdR)(Foulon et Al., 1996)

A mixture of IUdR (478 mg, 1.35 mmol), (dba)3Pd2 (41.75 mg,0.04 mmol) and bis(tributyltin) (1.95 g, 3.36 mmol, 1.7 mL) in DMF(5 mL) was heated at 65 1C for 5 h. The reaction mixture wasfiltered through celite and the solvent was removed by rotaryevaporator under reduced pressure. The crude oily product waspurified by silica gel chromatography (eluent: chloroform/methanol¼10/1) to afford 432.9 mg of TUdR (62%).

1H-NMR (CDCl3, 400 MHz): δ 7.22 (s, 1 H, H-6), 6.1 (t, J¼6.8 Hz,1 H, H-1′), 4.56 (m, 1 H, H-3′), 4.01 (m, 1 H, H-4′), 3.81(m, 2 H,H-5′), 2.45~2.28 (m, 2 H, H-2′), 1.49∼0.88 (m, 27 H, SnBu3); 13CNMR (CDCl3, 100 MHz): δ 166.3 (C, CQO), 151.2 (C, CQO), 145.1(CH, C-6), 113.1 (C, C-5), 87.7 (CH, C-3′), 87.1 (CH, C-1′), 71.8 (CH,C-4′), 62.5 (CH2, C-5′), 39.7 (CH2, C-2′), 28.9 (CH2, SnBu3), 27.2(CH2, SnBu3), 13.6 (CH3, SnBu3), 9.8 (CH2, SnBu3); ESI mass calcdfor C21H37N2O5Sn−, 517.17; found 517.13

2.5. Preparation of [131/123I]ICdR and [131/123I]IUdR

Starting from the organotin precursor TCdR and TUdR, no-carrier-added (n.c.a.) [131/123I]ICdR and [131/123I]IUdR were prepared(Scheme 2). To a 300-μL V-vial coated with 25 μg of TCdR andcontaining 20 μL ethanol and 3.7–37MBq of n.c.a. sodium iodide-131 (or iodide-123), 100 μL of oxidizing agent (H2O2:1 N HCl:H2O¼8:8:84) was added. The reaction mixture was vortexed inter-mittently. After 15 min, 100 μL sodium thiosulfate (1 M) and 100 μLsodium bicarbonate (saturated solution) was added to quench thereaction. The radiochemical purity of [131/123I]ICdR and [131/123I]IUdRproducts were determined using TLC and HPLC. TLC was performedon a reversed-phase C18 coated aluminum sheet (MERCK, Darmstadt,Germany), using 10 mM acetic acid/ethanol (2/1, v/v) as developingagent. The Rf value of [131I]-free iodide, [131I]ICdR and [131I]IUdR were0.98, 0.71 and 0.65, respectively (Fig. 1). Chromatograms wererecorded using an imaging scanner (system 200, Bioscan, USA). HPLCanalysis was performed on a reversed-phase column (RPR-1, Hamil-ton, USA) using acetonitrile/0.02 M acetic acid (10/90, v/v) as theeluent at a flow rate of 1 mL/min. The [131/123I]ICdR preparation wasidentified by co-injection with the previously characterized authenticICdR. Both authentic (5.72 min) and radioactive ICdR (6.11 min)compounds displayed consistent HPLC retention time (Fig. 2). Thedifference in the retention time is due to the physical separation

between the UV detector and the radiodetector. The [131/123I]ICdRsolution, eluted through a 0.22-μm apyrogenic disk, was ready forbiological studies. The radiochemical purities of [131/123I] ICdR and`[131/123I]IUdR were both ≧98%. The overall radiochemical yield wasabout 93%.

The specific activity of our [131I]ICdR product determined bystandard addition method (Brown and Reith, 1967) was 3.89�107 MBq/mmol.

2.6. Partition coefficient of [131I]ICdR

The partition coefficient of [131I]ICdR was determined bymeasuring the distribution of radioactivity in 1-octanol andphosphate buffered saline (PBS) and expressed as log P (Krisset al., 1963). [131I]ICdR (3.7 KBq) was added into a tube containing1 mL each of 1-octanol and PBS. After vortexing, the tube wascentrifuged for 5 min to ensure complete separation of layers.Then, 100 μL of each layer was collected into individual tubes, andmeasured by a γ-counter (1470 WIZARD Gamma Counter, Wallac,Finland). The measurement was repeated four times. Log P valueswere calculated using the following formula:

logP ¼ logðradioactivity in octanol layer=radioactivity in PBS layerÞ

2.7. Serum stability assays

The in vitro stability was evaluated by incubation of 3.7 MBq of[131I]ICdR and [131I]IUdR with mouse serum (0.5 mL) at 37 1C andexpressed by the radiochemical purity (RCP) at different timepoints (1, 2, 4, 8, and 24 h). The radiochemical purity wasdetermined by TLC method.

Fig. 1. TLC chromatogram of (a) [131I]ICdR and (b) [131I]IUdR preparation. TLC was performed on a reversed-phase C18 coated aluminum sheet, using 10 mM acetic acid/ethanol (2/1, v/v) as developing agent. The Rf value of [131I]ICdR and [131I]IUdR were 0.71 and 0.65, respectively.

Fig. 2. HPLC chromatogram of [131I]ICdR preparation. Quality control of the TCdRlabeling with radio-HPLC, using ACN/20 mM acetic acid (10/90, v/v) as the eluent ata flow rate of 1 mL/min. The retention time of [131I]ICdR was 6.11 min (radio peak,A), the same as that obtained from the authentic ICdR (5.72 min, UV peak, B).

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2.8. Cell cultures and tumor xenografts in mice

NG4TL4 sarcoma cells were cultured in α-Minimum EssentialMedium (MEM) (containing 10% fetal bovine serum, 100 U/mlpenicillin and 100 μg/ml streptomycin) at 37 1C in a humidified

atmosphere with 5% CO2. NG4TL4 cells (2�105) in 0.1 mL normalsaline were inoculated into the right flank of six-week-old FVB/Nfemale mouse under anesthesia to produce subcutaneous xeno-graft of NG4TL4 tumor. All studies were performed when thetumor were 0.8–1.0 cm in diameter. The animal experiments wereapproved by the Institutional Animal Care and Use Committee ofthe National Yang-Ming University (Taipei, Taiwan).

2.9. Cellular uptake of [131I]ICdR and [131I]IUdR

Cellular uptake study was performed following the literaturemethod (Tjuvajev et al., 1995). One million cells were seeded into15 cm2 dishes containing 14 mL of culture medium. After 24 h ofgrowth, the medium was replaced with fresh serum-free mediumcontaining radiotracer [131I]ICdR or [131I]IUdR (0.5~1 mCi/mL med-ium). At designated time points (1, 2, 4 and 8 h), cells wereharvested from the dishes using a scraper. Then, the cell suspen-sion was transferred to a 15 mL conical tube and centrifuged(3500 rpm) for 2 min. After centrifugation, 100 mL medium wascollected to a pre-weighed counting tube and the remaining wasdiscarded. The cell pellet was frozen with dry ice and collected toanother pre-weighed counting tube. The weight of cell pellet andmedium was measured and the radioactivity determined by a

W.-Y. Chang et al. / Applied Radiation and Isotopes 77 (2013) 166–173170

gamma scintillation counter and normalized to the weight. Thein vitro accumulation of radiotracers was expressed as cell-to-medium ratio.

2.10. Biodistribution study

The biodistributions of radiotracers ([131I]ICdR and [131I]IUdR)were performed in tumor-bearing mice on the 10th day aftertumor implantation. Mice were fasted for 8 h before biodistribu-tion studies. Radiotracer (3.070.1 MBq) was injected via the tailvein. The mice were sacrificed by cervical dislocation at designatedtime points (1, 2, 4 and 8 h). Tumors and fourteen other tissues(blood, heart, lung, liver, stomach, small intestine, large intestine,spleen, pancreas, kidney, bone, marrow, brain and muscle) wereexcised, washed, weighed and assayed for radioactivity with agamma scintillation counter. The uptake of radiotracer in tissues(in counts per minutes) was corrected for decay, normalized tosample weight, and expressed as the percentage of injected doseper gram of tissue (%ID/g), the tumor-to-blood and tumor-to-muscle accumulation ratios.

2.11. Planar gamma imaging studies

Planar γ-imaging was performed using a dual-head γ-camera(ECAM; Siemens) equipped with a pinhole collimator. A staticemission scan was acquired for 30 min at 1, 2, 4, and 8 h afterintravenous administration of 4.870.1 MBq of [123I]ICdR or [123I]IUdR. To estimate radioactivity concentration, regions of interest

Fig. 3. In vitro stability of 131I-ICdR and 131I-IUdR in mouse serum at 37 1C(n¼3).

Fig. 4. Cellular uptake of [131I]ICdR (A) and [131I]IUdR (B) in NG4TL4 cells up to 8 h of incmedium ratio (C/M)7S.D.

were drawn over the target tumors and tissues (i.e., muscle) andthe values were corrected by subtracting background levels ofradioactivity, which was measured in the remote areas away fromthe animal body. Tumor radioactivity concentration was normal-ized by that in the muscle and expressed as the tumor-to-muscleaccumulation ratio.

2.12. Metabolites assay

Normal female FVB/N mice were injected with 9.25 MBq of[131I]ICdR and [131I]IUdR through tail vein, and sacrificed bycervical dislocation at different time points (0.25, 1 and 2 h post-administration for [131I]ICdR; 5 and 15 min for [131I]IUdR). Bloodsamples were obtained by cardiac puncture and then centrifugedat 13,000 rpm for 10 min. After centrifugation, the supernatant(~300 μL) was transferred to an 1.5 mL eppendorf tube containing300 μL of ethanol, and centrifuged again to obtain serum (withoutproteins). Radioactive metabolites of [131I]ICdR and [131I]IUdR inserum and urine were assayed by TLC method under the sameconditions as those used for [131I]ICdR and [131I]IUdR analysis.

3. Results

3.1. Partition coefficient and serum stability of [131I]ICdR and [131I]IUdR

A log P value of −0.94, similar to that reported for IUdR (−0.95;Ghosh and Mitra, 1991), indicated that [131I]ICdR is considerablyhydrophilic. The percentage of intact [131I]ICdR in mouse serumafter 24 h incubation was greater than 90%, while that of [131I]IUdRwas less than 70% (Fig. 3). The result revealed a better serumstability of [131I]ICdR than [131I]IUdR.

3.2. Cellular uptake of [131I]ICdR and [131I]IUdR

The cellular uptake of [131I]ICdR and [131I]IUdR in NG4TL4sarcoma cells kept increasing with time (Fig. 4.). The cell-to-medium (C/M) ratio of [131I]ICdR and [131I]IUdR reached75.3773.23 and 476.38736.51 after 8 h incubation. The high C/M ratio implies that both tracers were proper substrates for thephosphorylation by host kinase in tumor cells. Although the meanaccumulation rate of [131I]ICdR (9.08 mL medium/g cells/h) in 8 hincubation was slower than that of [131I]IUdR (66.25 mL medium/gcells/h), both nucleoside radiotracers exhibited steadily increasedaccumulation in fast proliferating tumor cells.

ubation (n¼4, three independent experiments). All values are expressed as cell-to-

W.-Y. Chang et al. / Applied Radiation and Isotopes 77 (2013) 166–173 171

3.3. Biodistribution studies

The radioactivity distribution in NG4TL4 sarcoma-bearing miceafter administration of [131I]ICdR and [131I]IUdR were shown inTables 1 and 2. Significantly high uptake in organs with fast prolifera-tion (tumor, small intestine, and bone marrow) was clearly observedsoon after injection. The high radioactivity accumulation in stomach,especially post-injection of [131I]IUdR, can be explained by uptake offree 131I-iodide generated from the deiodination of [131I]IUdR, [131I]ICdR and other radioactive metabolites in vivo. For [131I]ICdR, thetumor uptake peaked at 4 h p.i. (4.0970.50 %ID/g), and then declinedslowly till 8 h p.i. (2.6670.10 %ID/g). The tumor-to-muscle (T/M) ratiokept increasing with time (4.78 at 1 h p.i.), and reached as high as29.56 at 8 h p.i. due to the persistent tumor retention and very lowmuscle activity. For [131I]IUdR, with its less metabolic stability in vivo,the tumor uptake peaked at the first hour (5.5170.42 %ID/g), thendeclined gradually till 8 h p.i. (2.3270.49 %ID/g). The tumor-to-muscle(T/M) ratio increased with time (4.55 at 1 h and 14.50 at 8 h p.i.), butto a lesser extent compared with [131I]ICdR. The very high radioactivityin urine and appreciable radioactivity found in kidney may suggestthat [131I]ICdR and [131I]IUdR and their metabolites mainly excrete viathe urinary system.

Table 1Radioactivity distribution in tumor and other normal tissues of NG4TL4 sarcoma-bearing FVB/n mice after intravenous injection of 3.070.1 MBq of [131I]ICdR.

Organ 1 h 2 h 4 h 8 h

Blood 3.8770.31 2.9170.35 2.2670.60 0.1670.14Heart 1.2170.13 1.2470.14 0.6670.54 0.0570.02Lung 1.5270.66 1.8170.16 1.4770.89 0.1370.06Liver 1.2670.15 1.4870.22 1.2670.88 0.1770.15Stomach 8.6270.99 7.7171.68 6.9171.43 0.6570.10S.Int 4.3972.66 5.1970.90 4.5771.67 3.4171.07L.Int 1.5970.17 1.3770.76 1.7470.56 0.4970.34Spleen 3.8870.40 3.7370.54 3.8170.69 1.7970.53Pancreas 1.7670.26 1.1970.88 1.3870.80 0.0970.03Kidney 2.7470.31 1.9670.22 1.6370.08 0.1970.06Muscle 0.770.34 0.5670.32 0.5170.34 0.0970.02Tumor 3.3570.02 3.6970.72 4.0970.50 2.6670.10Bone 0.7670.03 0.5170.12 0.4770.03 0.170.02Marrow 3.3070.06 3.6870.12 3.7270.34 2.7170.08Brain 0.1970.04 0.1570.03 0.1270.01 0.0170.00T/M 4.78 6.58 8.02 29.56T/B 0.86 1.26 1.81 16.6

Values were presented as %ID/g (mean7SD, n¼5 at each time point).

Table 2Radioactivity distribution in tumor and other normal tissues of NG4TL4 sarcoma-bearing FVB/n mice after intravenous injection of 3.070.1 MBq of 131I-IUdR.

Organ 1 h 2 h 4 h 8 h

Blood 3.6170.07 2.7770.73 1.8270.61 0.1170.07Heart 1.0170.07 0.9470.22 0.6870.16 0.1970.09Lung 1.8470.15 1.6270.43 1.3970.61 0.1270.02Liver 1.4070.18 1.5170.12 1.2370.41 0.1970.07Stomach 17.9472.17 13.5076.32 7.4971.69 0.9870.20S.Int 5.5570.94 4.8971.49 4.6671.38 3.4470.55L.Int 1.8270.43 1.5170.21 1.6670.33 1.9870.32Spleen 4.7170.75 5.3570.36 4.7070.25 2.4970.47Pancreas 2.3270.24 1.7670.45 1.7570.66 0.1670.04Kidney 2.7870.88 1.9870.60 1.9970.71 0.2870.10Muscle 1.2170.10 0.9070.12 0.7770.46 0.1670.02Tumor 5.5170.42 5.4970.56 4.8770.72 2.3270.49Bone 1.4970.41 1.2270.27 0.6270.31 0.1670.08Marrow 7.7672.51 6.8171.01 5.5973.84 4.1170.98Brain 0.3170.09 0.1970.01 0.1570.02 0.0270.01T/M 4.55 6.10 6.32 14.50T/B 1.52 1.98 2.67 21.09

Values were presented as %ID/g (mean7SD, n¼5 at each time point).

3.4. Gamma planar imaging

Sequential gamma planar imaging of NG4TL4 sarcoma-bearingmouse was performed after [123I]ICdR or [123I]IUdR injection. Theimages (Fig. 5) revealed significant tumor uptake at the first hourand persisted till 8 h post-injection. The T/M ratio derived fromthe images of [123I]ICdR was higher than that of [123I]IUdR in thewhole study period. The clearance of both radiotracers fromnormal tissues was obvious, most of the radioactivity diminishedat 8 h post-injection. Remarkable radioactivity retention in intes-tines, a known fast proliferation organ, was also noticed. Thebladder was clearly visible during the period of scintigraphicimaging, indicated that renal clearance is the predominant routefor the excretion of nucleoside radiotracers [123I]ICdR and [123I]IUdR and their radioactive metabolites. The results observed inscintigraphic imaging were consistent with those obtained frombiodistribution studies.

3.5. Metabolites assay

The radioactive components in the blood and urine of normalfemale FVB/N mice after injection of [131I]ICdR and [131I]IUdR wereanalyzed. For [131I]ICdR, the 131I-containing components in bloodwere 63.1% in [131I]ICdR, 18.6% in [131I]IUdR and 18.2% in 131I-iodideat 15 min p.i.. Until 120 min post-injection, the major radioactivecomponent in the blood and urine was [131I]ICdR (Table 3A). Theresults indicate that [131I]ICdR was metabolically stable in vivo. Onthe other hand, the degradation rate for [131I]IUdR was relativelyfaster. The 131I-containing components in blood were 25.2% in[131I]IUdR and 71.1% in 131I-iodide at 5 min p.i.. At 15 min post-injection, the major radioactive component is 131I-iodide in bothblood and urine (Table 3B).

4. Discussion

During the last two decades, several positron-emitting radionu-clide-labeled thymidine analogs, such as [18F]FLT (Shields et al., 1998;Vesselle et al., 2002), [18F]FMAU (Sun et al., 2005b; Tehrani et al., 2007)and [18F]FUdR (Kameyama et al., 1995; Wang et al., 2006), weredeveloped for non-invasive tumor detection and therapeutic efficacymonitoring with PET. However, the choice of gamma imaging probesfor tumor detection, especially specific for proliferation, is limited anddevelopment in this field receives much less attention. In this study,[131/123I]ICdR was evaluated as a SPECT probe for imaging tumorproliferation in vivo and compared with [131/123I]IUdR.

N.c.a. [131I]ICdR can be prepared using a simple method (fromTCdR) with high specific activity (3.89�107 MBq/mmol). Krisset al. has reported direct labeling of CdR with carrier-added 131I-ICl to obtain [131I]ICdR with low specific activity (7.05�102 MBq/mmol) (Kriss et al., 1962).

Thymidine kinase, especially TK1, is expressed in proliferatingcells, both normal and malignant (Kit, 1985). The relative activityof TK1 to pyrimidines was thymidine4deoxyuridine ⪢ deoxycy-tidine (Johansson and Eriksson, 1996). IUdR, a thymidine analog, isa better substrate of thymidine kinase than ICdR, a cytidine analog.In this study, the results that cellular uptake of [131I]IUdR was notonly higher but also faster than that of [131I]ICdR at every timepoints (Fig. 4), were consistent with those reported by Johanssonand Eriksson.

One of the important criteria for a PET or SPECT tracer is thein vivo stability. If it were degraded in the living subject, theradioactive metabolites should be rapidly excreted and not tointerfere with imaging and diagnosis (Toyohara et al., 2008). Inthis study, we have shown that [131I]ICdR was stable in vitro(Fig. 3) and degraded much more slowly compared with [131I]IUdR.

Fig. 5. The gamma planar images of FVB/N mice bearing NG4TL4 sarcoma (arrow head) after intravenous injection of 4.870.1 MBq of [123I]ICdR (A) and [123I]IUdR (B).Tumor-to-muscle (T/M) ratios were derived from the gamma planar images (n¼4).

Table 3Radioactive metabolites in blood (devoid of protein) and urine of FVB/N mice afterintravenous injection of [131I]ICdR (A) and [131I]IUdR (B).

(A)

Radioactive species 15 min 60 min 120 min

Blood[131I]ICdR (%) 63.1 72.1 67.7[131I]IUdR (%) 18.6 0 0[131I]iodide (%) 18.2 14.1 19.0[131I]compounda (%) 0 13.7 13.2

Urine[131I]ICdR (%) 56.4 71.0 81.1[131I]IUdR (%) 3.1 0 0[131I]iodide (%) 40.3 26.5 15.3[131I]compounda (%) 0 2.4 3.53

(B)

Radioactive species 5 min 15 min

Blood[131I]IUdR (%) 25.2 6.4[131I]IU (%) 3.6 1.5[131I]iodide (%) 71.1 92.0

Urine[131I]IUdR (%) 8.6 5.2[131I]IU (%) 4.4 2.7[131I]iodide (%) 86.9 92.0

a The compound is unknown.

W.-Y. Chang et al. / Applied Radiation and Isotopes 77 (2013) 166–173172

The metabolite assay showed that most [131I]ICdR remained intactin vivo and accounted for 72.2% of radioactivity in blood and 71.0%of radioactivity in urine at 60 min p.i.. (Table 3A). In addition, amarked portion of [131I]IUdR was observed in blood (18.6%) andurine (3.2%) at 15 min after administration of [131I]ICdR indicatingthat deamination of [131I]ICdR to produce [131I]IUdR proceeded at anoticeable rate in mouse. The metabolically produced [131I]IUdR isunstable in vivo and undergoes rapid deiodination, and thus noneof [131I]IUdR can be detected in blood or urine after 60 min p.i..Free 131I-iodide was formed due to the deiodination of [131I]ICdR

and [131I]IUdR in liver. The proportion of free 131I-iodide in urinedecreased with time, but that of [131I]ICdR grew in blood(Table 3A). The results might suggest that the free 131I-iodide inurine comes mainly from the deiodination of [131I]IUdR. [131I]IUdRis biologically unstable after i.v. injection in mice. At 5 min after i.v.administration, the major radioactive component is 131I-iodidein vivo and accounts for 71.1% radioactivity in blood and 88.0%radioactivity in urine (Table 3B), consistent with that reported byKassis (1996).

The biodistribution studies of [131I]ICdR and [131I]IUdR inNG4TL4 sarcoma-bearing mice revealed highly specific accumula-tions in fast proliferating tissues with active DNA synthesis (tumor,bone marrow, spleen and small intestine) than in low- or non-proliferating organs (brain, muscle, liver, and lungs) (Tables 1and 2). The tumor uptake of [131I]ICdR kept increasing up to 4 h p.i.owing to its high in vivo stability. For [131I]IUdR, with its muchpoor in vivo stability, the tumor uptake peaked at the first hourpost injection and then declined with time. The lower radioactivityin blood results in higher tumor-to-blood ratio post-injectionof [131I]IUdR (1.52, 1.98, 2.67 and 21.09 at 1, 2, 4 and 8 h p.i.)compared with that of [131I]ICdR (0.86, 1.26, 1.81 and 16.60 at 1, 2,4 and 8 h p.i.).

Gamma planar imaging of tumor-bearing mice after adminis-tration of [123I]ICdR and [123I]IUdR both showed significant radio-activity accumulation in the tumor and in tissues with rapidproliferation. The tumor lesion in the right hinder flank of mousecan be clearly delineated, and the tumor-to-muscle ratio increasedwith time. (Fig. 5) These results are consistent with those observedin biodistribution studies. Normal organs with low proliferationrate revealed low radioactivity retention at 8 p.i.. High radio-activity accumulation in bladder indicated the route of bodyclearance was predominantly via renal system.

5. Conclusion

In this study, radioiodine labeled no-carrier-added pyrimidineanalogs (n.c.a. [131/123I]ICdR and [131/123I]IUdR) were successfullyprepared. The results of biological characterizations demonstrated

W.-Y. Chang et al. / Applied Radiation and Isotopes 77 (2013) 166–173 173

that [123I]ICdR is a more promising probe than [123I]IUdR forevaluation of tissue proliferation in a sarcoma-bearing mousemodel. [123I]ICdR SPECT may provide information on tumorbiology and to monitor tumor response during and after cancertreatment in the future.

Acknowledgment

The authors thank the financial support from National Sciencecouncil, Taiwan (NSC95-2314-B010-090, NSC96-2113-M-010-002,NSC99-2314-B-010-027-MY3) and Cheng Hsin General Hospital,Taiwan (98F117CT07). The authors also appreciate the technicalsupport from National Research Program for Genetic Medicine,Taiwan (Molecular and Genetic Core, NSC100-2319-B-010-003).

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