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Applied Radiation and Isotopes 64 (2006) 342–347

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Synthesis and evaluation of a technetium-99m-labeleddiethylenetriaminepentaacetate–deoxyglucose complex

([99mTc]–DTPA–DG) as a potential imaging modality for tumors

Yue Chena,�, Zhan Wen Huanga, Ling Heb, Shi Long Zhengb, Ju Lian Lib, Da Lian Qinc

aDepartment of Nuclear Medicine, Affiliated Hospital, Luzhou Medical College, Luzhou, Sichuan 646000, PR ChinabWest China School of Pharmacy, Sichuan University, Chengdu, Sichuan 610041, PR China

cSchool of Pharmacy, Luzhou Medical College, Luzhou, Sichuan 646000, PR China

Received 23 July 2005; received in revised form 21 August 2005; accepted 22 August 2005

Abstract

This study describes the radiolabeling and preliminary biologic testing of diethylenetriaminepentaacetic acid (DTPA)–deoxyglucose

(DG) labeled with 99mTc. A one-step [99mTc]–DTPA–DG kit was prepared using the stannous chloride reduction method. When99mTcO4

� was added to the DTPA–DG kit at room temperature the radiochemical purity 30min later was 99.2%, and it remained

498.6% for 6 h. Rapid blood clearance of [99mTc]–DTPA–DG was observed in in vivo biodistribution, the main route of clearance was

via the kidneys. No significant accumulation in any other organs was seen. The tumor-to-brain and tumor-to-muscle concentration ratios

for [99mTc]–DTPA–DG uptake were higher than those for fluorine-18-flurodeoxyglucose (18F–FDG). Scintigraphic results demonstrated

the feasibility of [99mTc]–DTPA–DG imaging tumors. The [99mTc]–DTPA–DG complex is a potential imaging agent due to the ideal

physical characteristics of the radionuclide, ease of preparation, low cost, early accumulation and the preference for the renal route of

excretion.

r 2005 Elsevier Ltd. All rights reserved.

Keywords: [99mTc]–DTPA–DG; Tumor; Scintigraphic imaging; Radiopharmaceutical

1. Introduction

The accurate and early non-invasive detection ofmalignant disease is an important factor in the treatmentand prognosis of a cancer patient. Improvements in tumorradionuclide imaging depend on the development of moretumor-specific radiopharmaceuticals. Fluorine-18 –fluro-deoxyglucose ([18F]–FDG), a glucose analog, has becomethe preeminent radiopharmaceutical agents for the detec-tion of tumor tissue by positron emission tomography(PET), and the most commonly used PET tracer in clinicalpractice (Bar-Shalom et al., 2002; Kumar et al., 2003; Brinket al., 2004). High FDG tumor uptake is due to theincreased activity of hexokinase isoenzymes; thus FDG

e front matter r 2005 Elsevier Ltd. All rights reserved.

radiso.2005.08.004

ing author. Tel.: +86830 3162339; fax: +86 830 2392753.

ess: chenyue5523@126.com (Y. Chen).

reflects tumor cell proliferative activity as well as viability(Gatley, 2003).Because the production of 18F requires a cyclotron and

the isotope has a short (110min) half-life, its utility issomewhat limited compared to that of single photonemitters in nuclear medicine. By comparison, 99mTc, theisotope most commonly used in single emission-computedtomography (SPECT), is produced in the form ofNa99mTcO4 from a 99Mo generator; hence, it is widelyavailable and relatively inexpensive.Technetium-99m can easily be made to react with a wide

variety of hydrophilic compounds which include N4 (e.g.tetraazacyclododecane tetraacetic acid), N3S (e.g. merca-pato acetyl triglycine), N2S2 (e.g ethylenedicysteine diethy-lester), O4 (e.g. diethylenetriaminepentaacetic acid(DTPA)) and hydrazinenicotinamide chelates (Yang etal., 1999; Vera et al., 2001). Yang and Bayly et al have

ARTICLE IN PRESSY. Chen et al. / Applied Radiation and Isotopes 64 (2006) 342–347 343

developed a 99mTc labeled deoxyglucose (DG) analog(Yang et al., 2003; Yang et al., 2004; Bayly et al., 2004).

Diethylenetriaminepentaacetic acid can be labeled with99mTc easily, efficiently and with high radiochemical purityand stability. We previously reported on [99mTc]–DTPAchelators for use in functional imaging in oncology (Chenet al., 1999). We hypothesize that DTPA–deoxyglucose(DTPA–DG) labeled with 99mTc is a specific glucosetransport-targeted agent. If the binding of [99mTc]–DTPA–DG to tumor cells could be detected with a gammacamera, the tumor tissue could be imaged. This paperreports the synthesis and characterization of [99mTc]–DTPA–DG and demonstrates the feasibility of tumordetection.

2. Materials and methods

D-glucosamine hydrochloride was purchased from Al-drich Chemical Company (Milwaukee, WI); DTPA waspurchased from Sigma Chemical Company (St. Louis,MO). Most other chemicals were purchased from com-mercial suppliers and used as received. All aqueoussolutions were prepared using deionized water. The[18F]–FDG was purchased from the PET Center, People’sHospital of Sichuan Province, Chengdu, China.

2.1. Synthesis of DTPA–DG

Synthesis of condenses of DTPA with D-glucosamine: Ina hood, 10mL thionyl chloride was added dropwise to3.94 g of DTPA with efficient stirring at 0 1C, and thencontinuously stirred in boiling water for 3 h. Then themixture was refluxed for 20 h. The excess thionyl chloridewas distilled off in vacuo to give a yellowish powder, yield3.76 g. To a flask containing dianhydride acylchloride(3.76 g), dimethyl sulfoxide (30 cm3), pyridine (5 cm3) andD-glucosamine hydrochloride (6.47 g) were added, and themixture was stirred in a boiling water bath for 24–48 h.Then the mixture was separated by dialysis or sephadexG15 with water as eluent to give DTPA–DG.

2.2. 99mTc labeling of DTPA–DG.

Tin chloride reduction was used to label DTPA-DG with99mTc in DTPA–DG kits prepared to contain 25mgDTPA–DG and 0.5mg SnCl2 in 1–3 cm3 solution. The pHwas subsequently adjusted to 6.0. The mixture was passedthrough a 0.22mmMillipore membrane filter into a sterile vial.

In a kit vial 200–300MBq 99mTcO4� in 1–2mL isotonic

saline, obtained from a commercial 99Mo-99mTc generator,was added, gently mixed and incubated at room tempera-ture for 30min. The resulting solution was used within 6 hafter preparation. The radiochemical purity was determinedby thin layer chromatography. The sheets of No. 1 Xinhuafilter paper (Hangzhou Xinhua Group Co., Ltd., Hangzhou,China) were cut into 1.2� 30 cm strips. The strips weredeveloped with the eluent of acetone and 0.9% saline

solution as the mobile phase. The strips were cut afterelution as appropriate and counted with a gamma-wellcounter. Pertechnetate moved with the solvent front andreduced hydrolyzed 99mTc remained at the origin in bothsolvents; [99mTc]–DTPA–DG remained at the origin inacetone, but moved with the solvent front in 0.9% salinesolution. The radiochemical purity was determined bysubtracting the sum of the amounts that migrated in acetoneand that which remained at the origin in saline from 100%.The stability of [99m]–DTPA–DG was tested at 0.5, 1, 3 and6 h after storage at room temperature using same method.

2.3. Cellular uptake assay in vitro

Cellular uptake assay in vitro was performed in multi-well plates using a human mammary cancer cell line (MCF-7). To each well (n ¼ 18) containing 80,000 cells was added0.074MBq of [99mTc]–DTPA–DG to the first group of sixwells; 0.074MBq of [18F]–FDG to the second group of sixwells and 0.074MBq of [99mTc]–DTPA to a control groupof six wells. After incubation for 0.5–4.0 h at 37 1C, the cellswere washed three times with phosphate-buffered salineand then once with trypsin to form the cell suspensions andto remove them with a radiotracer uptake. The cellsuspensions were placed in a gamma counter (Ri HuanInstrument Factory of Shanghai) to determine the amountof cell-associated radiotracer. The percentage uptake of thetotal activity was calculated.

2.4. Biodistribution in vivo

The animals were housed at The Animal Center ofLuzhou Medical College. A total of 40 female nude rats(Laboratory Animal Center of Sichuan University) wereused . Each animal was xenografted in the right forelegmuscle with 3� l06 cells of the MCF-7 human mammarycancer cell line (The Key Laboratory of Biotherapy ofHuman Disease of Ministry of Education, West ChinaHospital, Sichuan University). Two weeks after grafting, thetumors had attained a size of about 6mm in diameter. Twoseparate biodistribution studies with [99mTc]–DTPA–DG(group 1) and [18F]–FDG (group 2) were conducted. Eachgroup of rats received [99mTc]–DTPA–DG or [18F]–FDGintravenously. The injected activity was 0.037–0.111MBqper mouse. They were killed by decapitation for the[99mTc]–DTPA–DG group at 10min, 1, 2, 4 and 8h, andfor the [18F]–FDG group at 10min, 2 and 4h. After sacrifice,selected tissues were excised, weighed and the radioactivitywas measured. The biodistribution of radiotracer in eachtissue sample was calculated as percentage injected dose pergram of wet tissue. Tumor-to-non-target tissue ratios werecalculated from the corresponding tissue concentrations.

2.5. [99mTc]–DTPA–DG imaging

After the intravenous injection of 11.1MBq[99mTc]–DTPA–DG in 0.1 cm3 into three rats bearing

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Fig. 2. In vitro cellular uptake of [99mTc]–DTPA , [99mTc]–DTPA–DG

and [18F]–FDG. There was an obviously increased uptake of

[99mTc]–DTPA–DG and [18F]–FDG compared with the uptake of

[99mTc]–DTPA as a function of time. Data are expressed as % uptake

(mean with SD). * Po0:05 vs. [18F]–FDG by Student pair t-test.

Y. Chen et al. / Applied Radiation and Isotopes 64 (2006) 342–347344

mammary tumor xenografts, anterior and posterior wholebody scans were obtained with a dual-head gamma(Millennium VG5, GE Medical Systems, Milwaukee,USA) equipped with a parallel-hole, low-energy, all-purpose collimator. Images were recorded at 0.5, 2.0 and4.0 h after injection. The ROI between tumor tissue andcontralateral thigh muscle (background) was used todetermine tumor-to-background ratios. To ascertainwhether tumor uptake of [ 99mTc]–DTPA–DG was perfu-sion related, images were also recorded with [99mTc]–DTPA in the mammary tumor-bearing rats at 0.5, 2.0 and4.0 h after 11.1MBq of the agent was injected.

2.6. Statistical analysis

The in vitro percentage of cellular uptake, in vivopercentage of injected dose per gram of wet tissue weightand tumor-to-non-tumorous tissue ratios are presentedas means7standard deviations. To compare differences inpercentage of cellular uptake between the [99mTc]–DTPA–DG and 18F–FDG groups, the Student t-test wasused for comparisons between groups. P values o0.05were considered a statistically significant difference.

3. Results

3.1. Chemical structure and 99mTc labeling of DTPA–DG

The chemical structure of DTPA–DG is shown in Fig. 1.The radiochemical purity of [99mTc]–DTPA–DG reached99.2% 30min after 99mTcO4

� was added to a DTPA–DGkit at room temperature. The radiochemical purityremained 498.6% for 6 h after labeling. The shelf life ofthe kit lasts at least 3months in a refrigerator at 4 1C.

3.2. Cellular uptake in vitro

There was a significantly increased uptake of[99mTc]–DTPA–DG and [18F]–FDG in mammary cancercells as a function of time compared with the uptake of[99mTc]–DTPA (Po0:05). Maximal uptake of [99mTc]–

Fig. 1. Chemical structure of DTPA–DG

DTPA–DG occurred at 4 h after injection, and reached alevel of 0.5% administered activity, whereas [18F]–FDGhad a 40.6% uptake at 4 h after incubation (Fig. 2).

3.3. Biodistribution in rats

Rapid blood clearance of [99mTc]–DTPA–DG wasobserved with the main route of clearance via the kidneys.No significant accumulation in any other organs was seen.In group 1, [99mTc]–DTPA–DG had higher tumor-to-muscle tissue and tumor-to-brain tissue ratios as afunction of time, whereas in group 2, [18F]–FDG hadhigher tumor-to-blood ratios (Tables 1 and 2).

3.4. [99mTc]–DTPA–DG imaging

There was a marked increase in uptake, as demonstratedby the ROI ratios of tumor versus non-tumorous uptakeas a function of time. Scintigrams demonstrated thathuman breast tumor xenografts in rats could be bettervisualized with [99mTc]–DTPA–DG scintigraphy thanwith [99mTc]–DTPA scintigraphy at the same time points(Fig. 3a). The ROI ratios of [99mTc]–DTPA–DG uptakedetermined 0.5 and 2 h after radionuclide administrationfor the tumors versus the corresponding non-tumor regions(T/NT ratios) were 2.4671.02 and 3.5471.36, respectively.The kidneys, liver and bladder were visualized (Fig. 3b). Inthe [99mTc]–DTPA control group, the T/NT ratios were1.1670.02 (po0:01) and 1.1470.03 (po0:01) at 0.5 and2 h, respectively.

4. Discussion

A simple, rapid and easy procedure for the preparationof highly pure [99mTc]–DTPA–DG that can be used forimaging tumor tissue has been developed. The findings ofthe biodistribution and imaging with [99mTc]–DTPA–DG

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Table 1

Biodistribution of 99mTc–DTPA–DG in nude rats bearing MCF-7 human mammary tumour xenografts

Organ Time after injection

10min 1 h 2 h 4 h 8 h

Blood 11.3870.9 2.4070.73 0.6770.18 0.4970.11 0.570.08

Heart 4.9870.61 0.9970.28 0.2970.03 0.3270.10 0.2570.04

Lung 9.1372.30 1.7270.33 0.6870.07 0.4270.12 0.3370.16

Liver 5.2070.93 2.6870.32 2.7671.05 1.6770.29 1.3770.47

Spleen 7.5570.94 5.0671.13 5.8770.60 6.3971.55 2.6970.63

Kidney 28.8678.88 10.6374.35 4.4570.98 4.2272.00 1.9970.12

Stomach 5.9873.49 2.2872.23 1.7670.84 0.4070.08 0.4770.15

Intestine 3.4971.34 1.4771.12 1.3071.07 0.3070.12 0.2470.03

Muscle 4.3471.96 1.1870.71 0.4270.17 0.3770.22 0.3570.18

Brain 0.5170.09 0.2270.11 0.0970.02 0.0870.02 0.0670.01

Tumour 5.1271.43 3.1070.87 2.1070.02 1.5970.04 1.6970.03

Tumour/Blood 0.4570.09 1.2970.26 3.1370.63 3.2470.65 3.3870.68

Tumour/Muscle 1.1870.24 2.6370.53 5.0171.02 4.3070.89 4.8370.97

Tumour/Brain 10.0475.89 14.0977.91 23.3376.61 19.8873.45 28.1776.21

% Injected activity/g, mean7SD, n ¼ 3.

Table 2

Biodistribution of 18F–FDG in nude rats bearing MCF-7 human

mammary tumour xenografts

Organ Time after injection

10min 2 h 4 h

Blood 1.9270.65 0.2570.02 0.2070.02.

Heart 11.8276.24 16.3977.43 18.3376.61

Lung 2.4371.01 2.2270.98 2.3171.02

Liver 2.0671.05 0.6070.23 0.7470.21

Spleen 2.6771.03 1.6170.12 1.8470.41

Kidney 1.7271.01 0.8770.12 1.3270.08

Stomach 4.6571.68 4.4571.23 2.5671.02

Intestine 2.3771.25 2.3971.01 1.3770.37

Muscle 4.3171.64 5.1572.36 4.1572.24

Brain 6.0772.33 3.0471.22 2.2470.97

Tumour 2.8471.03 1.4370.65 1.4270.12

Tumour/Blood 1.5871.65 5.7372.78 7.1272.12

Tumour/

Muscle

0.6870.25 0.2870.05 0.3670.06

Tumour/Brain 0.4970.18 0.4670.12 0.6570.09

% Injected activity/g, mean7SD, n ¼ 3:

Y. Chen et al. / Applied Radiation and Isotopes 64 (2006) 342–347 345

in the mammary tumor-bearing rats appear to support ourinitial hypothesis that [99mTc]–DTPA–DG has the poten-tial to be used as a functional imaging agent.

In addition, the uptake of [99mTc]–DTPA–DG inmammary tumor cell lines was comparable with that of[18F]–FDG, suggesting that the uptake of both imagingagents reflects the increased metabolism in proliferatingtumor cells. It has been confirmed that there are twomechanisms for the cellular processes of glucosamine atleast. The first mechanism resembles the cellular processmechanism of a glucose transporter system. In the secondmechanism, glucosamine enters cells and forms glucosa-mine-6-phosphate directly in the additional transcriptional

pathways (Yang et al., 2003; Bayly et al., 2004; Sasajima etal., 2004; Maher et al., 2005; Toyama et al., 2004; Jones etal., 2002; Carnochan and Brooks, 1999). The mechanism of[99mTc]–DTPA–DG is not yet understood but it may reflectmore signaling biosynthetic pathways than [18F]–FDG.Over expression of different glucose transporter modes

may lead to higher or lower detection specificity forradionuclide imaging. [99mTc]–DTPA–DG showed anadvantage over [18F]–FDG with regard to lower tumor-to-brain tissue and tumor-to-muscle tissue ratios; however,[18F]–FDG had higher tumor-to-blood ratios. Lowertumor-to-blood ratios of [99mTc]–DTPA–DG are causedby the higher concentration of this labeling agent in bloodcompared with that of [18F]–FDG in blood. The resultssuggest that [99mTc]–DTPA–DG can result in an increasein circulation time flow to the kidneys, and excretion, since[99mTc]–DTPA–DG showed lower uptake in the tumor,brain and heart than [18F]–FDG. The lower uptake of[99mTc]–DTPA–DG by normal brain tissue may be due tocoordination chemistry factors. The [99mTc]–DTPA–DG isa non-lipophilic radiopharmaceutical and incapable ofpassage of the blood–brain barrier (BBB) unless the barrieris disrupted. The 99mTc atom is stabilized by electrons fromthe nitrogens in the DTPA–DG molecule; hence, thecharge on the [99mTc]–DTPA–DG may reduce uptakeacross the BBB and in healthy brain cells. Thus, it ispossible that brain tumor cells may accumulate[99mTc]–DTPA–DG because of the disrupted BBB. Incontrast, 18F–FDG is a neutral molecule and can cross theBBB easily. Thus, [18F]–FDG PET may not be as effectivein the detection of low-grade tumors compared with uptakein normal gray matter. Because of the high tumor-to-background ratios demonstrated with [99mTc]–DTPA–DGimaging, this scan may be more suitable and effective in thediagnosis of low-grade brain tumors than [18F]–FDG PET(Yang et al., 2003; Toyama et al., 2004).

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Fig. 3. Planar imaging obtained at 2 h after administration of [99mTc]–DTPA (a) and [99mTc]–DTPA–DG (b) in a nude rat bearing the MCF-7 human

mammary cancer cells. The tumor could be better imaged by [99mTc]–DTPA–DG scintigraphy.

Y. Chen et al. / Applied Radiation and Isotopes 64 (2006) 342–347346

The feasibility of performing imaging with [99mTc]–DTPA–DG was evaluated in mammary tumor-bearing ratswith tumors in the foreleg. The agent was compared with[99mTc]–DTPA, and enabled good visualization of thetumors at 2 and 4 h after administration. [99mTc]–DTPA isa blood flow agent that has no target for any tissue;[99mTc]–DTPA was delivered to the liver and kidneysbecause of high levels of blood flow through these organs.The readily available [99mTc]–DTPA–DG enabled detec-tion of tumor tissues up to 2 and 4 h after injection whileremaining stable, as evidenced by the low uptake in thethyroid gland and stomach in the in vivo biodistributionand imaging, which suggested in vivo stability.

5. Conclusion

There are similarities between the uptake of [99mTc]–DTPA–DG and the uptake of 18F–FDG in tumors.Considering the advantage of obtaining 99mTc from agenerator system, cost effectiveness, easy availability andstability of [99mTc]–DTPA–DG, [99mTc]–DTPA–DG couldbe a potential imaging agent in the detection of tumor.

Acknowledgements

This research was supported by Educational Ministry ofSichuan Grant 2004B005 and Luzhou Medical CollegeResearch Program Grant 20040056. The authors are

thankful to Prof. Kuang An Ren for support andencouragement.

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