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DOI: 10.1148/radiol.2262011811
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(Radiology 2003;226:465-473.)
© RSNA, 2003

Experimental Studies

Imaging with 99mTc ECDG Targeted at the Multifunctional Glucose Transport System: Feasibility Study with Rodents1

David J. Yang, PhD, Chang-Guhn Kim, MD, Naomi R. Schechter, MD, Ali Azhdarinia, MS, Dong-Fang Yu, MS, Chang-Sok Oh, PhD, Jerry L. Bryant, MS, Jong-Jin Won, MD, E. Edmund Kim, MD and Donald A. Podoloff, MD

1 From the Divisions of Diagnostic Imaging (D.J.Y., C.G.K., A.A., D.F.Y., C.S.O., J.L.B., J.J.W., E.E.K., D.A.P.) and Radiation Oncology (N.R.S.), University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030. From the 2001 RSNA scientific assembly. Received November 9, 2001; revision requested January 7, 2002; final revision received May 16; accepted June 18. The animal research and nuclear magnetic resonance facility used in this study was supported by M.D. Anderson Cancer Center (Cancer Center Support Grant) grant NIH CA-16672. Supported in part by Cell Point Research Fund. Address correspondence to D.J.Y. (e-mail: dyang@di.mdacc.tmc.edu).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
PURPOSE: To evaluate the feasibility of technetium 99m (99mTc) ethylenedicysteine–deoxyglucose (ECDG) imaging in tumor-bearing rodents.

MATERIALS AND METHODS: ECDG was synthesized by means of reacting ethylenedicysteine with glucosamine, with carbodiimide as the coupling agent. Hexokinase assays were performed at an ultraviolet wavelength of 340 nm. To determine whether blood glucose level could be altered, ECDG or glucosamine was injected into six rats. In a separate study, ECDG followed by insulin was administered to three rats. To determine biodistribution, lung tumor cells were intramuscularly injected into the hind legs of 18 nude mice. The animals were then injected with 99mTc ECDG or fluorine 18 (18F) fluorodeoxyglucose (FDG) (0.037–0.074 MBq per mouse). Radioactivity was measured in tissue excised from the animals. Scintigraphy was performed in three groups: in group 1 to demonstrate that different-sized tumors could be imaged after 99mTc ECDG administration, in group 2 to ascertain whether tumor uptake of 99mTc ECDG was perfusion related, and in group 3 to demonstrate that tumor uptake of 99mTc ECDG occurred by means of a glucose-mediated process.

RESULTS: ECDG was positive for phosphorylation at hexokinase assay. Blood glucose level increased with ECDG injection and decreased with insulin administration. Tumor-to–brain tissue and tumor-to–muscle tissue ratios of 99mTc ECDG uptake were higher than those of 18F FDG uptake. Scintigraphic results demonstrated the feasibility of 99mTc ECDG imaging.

CONCLUSION: There are similarities between 99mTc ECDG uptake and 18F FDG uptake in tumors, and study findings supported the potential use of 99mTc ECDG as a functional imaging agent.

© RSNA, 2003

Index terms: Contrast media, experimental studies • Experimental study • Neoplasms, experimental studies • Radionuclide imaging, experimental studies


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
Improvements in tumor scintigraphy are fundamentally dependent on the development of more tumor-specific radiopharmaceutical agents. Owing to greater tumor specificity, radiolabeled ligands and radiolabeled antibodies have led to a new era in the scintigraphic detection of tumors and undergone extensive preclinical development and evaluation. Radionuclide imaging modalities such as positron emission tomography (PET) and single photon emission computed tomography (CT) are diagnostic cross-sectional imaging techniques that enable one to map the location and concentration of radionuclide-labeled compounds (1–3). Although CT and magnetic resonance (MR) imaging yield considerable anatomic information about the location and extent of tumors, these modalities do not enable one to adequately differentiate residual or recurrent tumors from edema, radiation necrosis, or gliosis. PET and single photon emission CT can be used to localize and characterize active tumors by enabling measurement of metabolic activity.

Fluorine 18 (18F) fluorodeoxyglucose (FDG) has been used to diagnose and stage tumors (4–14), myocardial infarction (15), and neurologic disease (16,17). Although tumor metabolic imaging with 18F FDG has been studied for more than 2 decades, the use of this examination in clinical practice is still limited by factors such as difficult access, limited availability, and high cost (18). In addition, PET radiosynthesis must be performed rapidly because of the short half-life of the positron isotopes. 18F chemistry studies are usually complicated and involve longer synthesis time (eg, 1 hour with FDG). Thus, it would be desirable to develop a simple technique to label agents with less costly isotopes for tissue-specific targeted imaging.

Technetium 99m (99mTc) has been preferred for labeling radiopharmaceutical agents owing to the low energy (140 Kev vs 511 Kev with 18F) and inexpensive isotope cost ($0.21/MBq vs $50/MBq for 18F) associated with the use of this element. Several 99mTc-labeled agents have been reported on; these include N4 (eg, tetraazacyclododecane tetraacetic acid), N3S (eg, mercapto acetyl triglycine), N2S2 (eg, ethylenedicysteine diethylester), NS3,S4 (eg, sulfur colloid), O4 (eg, diethylenetriaminepentaacetic acid), and hydrazinenicotinamide chelates (19–24).

Diethylenetriaminepentaacetic acid does not chelate with 99mTc with stability that is comparable to that when it chelates with indium 111, however. Imaging with 99mTc-labeled hydrazinenicotinamide requires two additional chemicals, tricine and triphenylphosphine, to form a 99mTc complex and thus is inconvenient and costly. The nitrogen and sulfur combination, however, has been shown to be a stable chelate for 99mTc. In addition, bis-aminoethanethiol tetradentate ligands, which are also known as diaminodithiol compounds, are known to form very stable technetium (valent V) oxide complexes owing to efficient binding of the oxotechnetium group to two thiolsulfur and two amine nitrogen atoms. 99mTc ethylenedicysteine is the most recent and successfully used N2S2 chelate (25,26). Ethylenedicysteine can be labeled with 99mTc easily, efficiently, and with high radiochemical purity and stability.

We previously reported on a series of 99mTc ethylenedicysteine conjugates for use in functional imaging in oncology (27–30). We hypothesize that 99mTc ethylenedicysteine–deoxyglucose (ECDG) is a specific multifunctional glucose transport–targeted agent. If the binding of 99mTc ECDG to tumor cells could be detected at planar scintigraphy or single photon emission CT, this depiction capability would suggest that the degree of malignancy (eg, tumor stage or tumor burden) of tumor cells could be imaged. Thus, the purpose of our study was to evaluate the feasibility of 99mTc ECDG imaging in tumor-bearing rodents.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
Chemicals and Analysis
Mass spectral analyses were conducted at the University of Texas Health Science Center in Houston, Texas. Nuclear MR spectra were recorded on a spectrometer (Bruker 500; Bruker Biospin, Rheinstetten, Germany). The mass data were obtained by means of fast atom bombardment (Kratos Mass Spectrometry 50; Kratos Analytical, Manchester, England). N-hydroxysulfosuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride were purchased from Pierce Chemical in Rockford, Illinois. Most other chemicals were purchased from Aldrich Chemical in Milwaukee, Wisconsin. 18F FDG was purchased from the Positron Diagnostic Research Center of the University of Texas. Silica gel–coated thin-layer chromatography plates were purchased from Whatman in Clifton, New Jersey.

Synthesis of Ethylenedicysteine
Ethylenedicysteine was prepared in a two-step synthesis process according to methods described by Blondeau et al (25) and Ratner and Clarke (26). Briefly, cysteine hydrochloride (41.52 g) was dissolved in 106 mL of water. Formaldehyde (26.1 mL) was added to the cysteine hydrochloride solution, and the reaction mixture was stirred overnight at room temperature. Pyridine (26.6 mL) was then added to the solution, and the precipitate formed. The crystals were separated and washed with 54 mL of ethanol for 25 minutes at room temperature and then filtered with a Buchner funnel (Alken-Murray, New Hyde Park, NY). The crystals were triturated with 150 mL of petroleum ether, filtered again, and then lyophilized for 3 days. The precursor, L-thiazolidine-4-carboxylic acid (melting point, 195°; reported 196°C to 197°C), was used for synthesis of ethylenedicysteine. The precursor (22 g) was dissolved in 200 mL of liquid ammonia and refluxed. Sodium metal was added until the agent had a permanent blue color. Ammonium chloride was added to the blue solution, and then the solvents were evaporated until they were dry. The residue was dissolved in 200 mL of water, and the pH was adjusted to 2. The precipitate that formed was filtered and washed with 500 mL of water. The solid material was dried in a calcium chloride vacuum dessicator (Aldrich Chemical, Milwaukee, Wis). Ethylenedicysteine was then prepared (melting point, 243°C to 246°C; reported 252°C to 253°C).

Synthesis of ECDG
Sodium hydroxide (concentration, 1 N; volume, 1 mL) was added to a stirred solution of ethylenedicysteine (concentration, 110 mg; volume, 0.41 mmol) in water (5 mL). To this colorless solution, N-hydroxysulfosuccinimide (concentration, 241.6 mg; volume, 1.12 mmol) and carbodiimide hydrocloride (concentration, 218.8 mg; volume, 1.15 mmol) were added. D-glucosamine hydrochloride salt (concentration, 356.8 mg; volume, 1.65 mmol) was then added. The solution was stirred at room temperature for 24 hours, and the pH was adjusted to 6.4–7.0. This solution was dialyzed for 48 hours by using a molecular porous membrane with a cutoff at 500 (Spectra/POR; Spectrum Medical Industries, Houston, Tex). After dialysis, the product was freeze dried with a lyophilizer (Labconco, Kansas City, Mo). The product weighed 291 mg (yield 60%). The product was depicted at proton nuclear MR imaging as follows: (D2O) {delta} 2.60–2.90 (multiple signals, 4H and -CH2-SH of ethylenedicysteine), 2.95 (triple signals, 2H, glucosamine 5-CH-CH2OH), 3.20 (double signals, 4H, glucosamine 6-CH2OH), 3.30–3.95 (multiple signals, 6H glucosamine 1,3,4-CH and 4H CH2-SH of ethylenedicysteine), 3.30–3.66 (multiple signals, 4H, CH2-CH2- of ethylenedicysteine), 4.15–4.30 (triple signals, 2H, NH-CH-CO of ethylenedicysteine), 4.60 (double signals, 2H, glucosamine 2-CH-NH2), where {delta} is the chemical shift. The fast atom bombardment mass spectrometry molecular weight was 591 (parent ion M+, 20).

Pertechnetate was obtained from Syncor Pharmaceutical in Houston, Texas. Radiosynthesis of 99mTc ECDG was achieved by means of adding the required amount of ECDG (80–100 mg) and tin (II) chloride (100 µg) to the pertechnetate. Radiochemical purity was assessed at radio–thin-layer chromatography (Bioscan, Washington, DC), with 1 mol/L of ammonium acetate plus methanol (4:1) as the eluant. High-performance liquid chromatography with a sodium iodide detector and ultraviolet detector (254 nm) was performed on a gel permeation column (Biosep SEC-S3000, 7.8 x 300 mm; Phenomenex, Torrance, Calif) at a flow rate of 1.0 mL/min. The eluant was 0.1% lithium bromide in phosphate-buffered saline (10 mmol/L, pH = 7.4).

Hexokinase Assay
To determine if ECDG mimics glucose phosphorylation (31), a hexokinase assay was performed. By using a ready-made assay kit (Sigma Chemical, St Louis, Mo), 1.0 mg of ECDG, 1.0 mg of FDG, and 1.0 mg of D-glucosamine each were dissolved in 1 mL of water separately, and 2.5 mg of D-glucose was dissolved in 2.5 mL of water. Next, 200 µL was removed from each of the four solutions and diluted in 2.5 mL of water. A 10-µL aliquot of each solution was removed, mixed with 900 µL of glucose reagent (Infinity Glucose Reagent; Sigma Chemical), and then incubated at 37°C for 3 minutes. The reduced-form of nicotinamide adenine dinucleotide was then assayed at an ultraviolet wavelength of 340 nm.

In Vitro Cellular Uptake Assay
In vitro cellular uptake assay was performed by using a human lung cancer cell line, A549. Each well (n = 12) that contained 80,000 cells was added to 0.074 MBq of 99mTc ECDG (in one group of six wells) and to 0.074 MBq of 18F FDG (in another group of six wells). After the cells were incubated for 0.5–4.0 hours, we washed them with phosphate-buffered saline three times and then with trypsin one time to remove some of the cells with radiotracer uptake. To evaluate if the uptake of 99mTc ECDG in cells is mediated by means of a D-glucose mechanism, we added 1.0 mg of D-glucose, 2.0 mg of L-glucose, and 0.074 MBq of 99mTc ECDG to each well that contained lung cancer cells (50,000 cells per 0.5 mL of solution per well). After the cells were incubated for 2 hours, we washed them with phosphate-buffered saline three times and then with trypsin one time to remove some of the cells with radiotracer uptake. The cells were counted by a gamma counter (Packard Instrument, Downers Grove, Ill).

Effect of ECDG Loading on Blood Glucose Level
The animals were housed at The University of Texas M.D. Anderson Cancer Center. All protocols involving animals (eg, rats and mice) were approved by the M.D. Anderson Animal Use and Care Committee. To determine whether the blood glucose level could be increased by administering either glucosamine or ECDG and decreased by administering insulin, we deprived nine healthy Fischer 344 rats (Harlan, Indianapolis, Ind) (three rats per agent), which weighed 145–155 g, of food overnight prior to the experiments. The concentrations of prepared glucosamine hydrochloride and ECDG were 60% and 164% (milligram percentage), respectively. Blood glucose level (in milligrams per deciliter [SI: millimoles per liter]) was measured by using a glucose meter (Glucometer DEX; Bayer, Elkhart, Ind). The baseline blood glucose level was determined prior to the study. Each rat (n = 6) was injected with 1.2 mmol of glucosamine (group 1, n = 3) or ECDG (group 2, n = 3) per kilogram of body weight.

In a separate experiment, three rats (group 3) were injected with ECDG and then with insulin (3 units) 30 minutes later. Blood samples from the tail vein were collected (D.F.Y.) every 30 minutes up to 6 hours after agent administration. The total numbers of rats examined in each experiment are listed in Table 1.


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  		TABLE 1. Summary of Rodent Studies

 
Tissue Distribution Studies with 99mTc ECDG
One author (D.F.Y.) intramuscularly injected A549 human lung cancer cells (3 x 106 cells per mouse) into the middle dorsal region in a total of 18 female athymic nude mice (Ncr-nu/nu; National Cancer Institute, Bethesda, Md). After the tumors reached a size of 6 mm in diameter, two separate biodistribution studies with 99mTc ECDG (group 1) and 18F FDG (group 2) were conducted. Each group of nine mice received 99mTc ECDG or 18F FDG intravenously. The injection activity per mouse was 1–3 µCi (0.037–0.111 MBq) per mouse. The injected amount of 99mTc ECDG was 0.2 mg per mouse.

In both groups of nine mice each, the animals were divided into three groups: The animals in the first group were sacrificed 0.5 hour after radiotracer administration; those in the second group, 2 hours afterward; and those in the third group, 4 hours afterward. After the rodents were sacrificed, selected tissues were excised and weighed, and the radioactivity was measured. The biodistribution of radiotracer in each tissue sample was calculated as a percentage of the injected dose per gram of wet tissue weight. Tumor-to–nontargeted tissue ratios were calculated from the corresponding values of injected dose per gram of wet tissue weight.

Gamma Scintigraphy Studies
The animal model used was that of breast tumor–bearing rats, because their size is suitable for imaging. One author (D.F.Y.) inoculated female Fischer 344 rats that weighed 250–275 g with mammary tumor cells from the 13762 tumor cell line (subcutaneous, 106 cells per rat). This tumor cell line is specific to Fischer rats. After 8–10 days, tumor volumes of 0.3–0.6 cm were measured. Scintigrams were obtained 0.5, 2.0, and 4.0 hours after the intravenous injection of 300 µCi (11.1 MBq) of either 99mTc ethylenedicysteine (in three rats) or 99mTc ECDG (in three other rats). To demonstrate that different-sized tumors could be imaged, an author (D.F.Y.) performed imaging with 99mTc ECDG in two rat groups: three animals with small tumors and three with medium-sized tumors. We analyzed the whole-body images by defining regions of interest (ROIs, in counts per pixel), which were essentially outlines of particular organs. The ROI between tumor tissue and muscle (at an symmetric site) was used to determine tumor-to–nontumorous tissue ratios.

To ascertain whether tumor uptake of 99mTc ECDG was perfusion related, one author (A.A.) performed planar scintigraphy with 99mTc ethylenedicysteine and 99mTc ECDG in the breast tumor–bearing rats; 300 µCi (11.1 MBq) of the agent was intravenously administered to each rat.

To demonstrate that tumor uptake of 99mTc ECDG occurred by means of a glucose-mediated process, six tumor-bearing rats were pretreated with 200 mg/kg of saline (three rats) or 200 mg/kg of intravenously administered unlabeled FDG (three rats) and then injected with 99mTc ECDG (11.1 MBq) 30 minutes later. In a separate experiment, three rats were pretreated with 3 units of intramuscularly administered insulin and then injected with 99mTc ECDG 30 minutes later. The total numbers of rodents examined in each study are listed in Table 1. The tumor-to–nontumorous tissue ratios in the FDG–99mTc ECDG and insulin–99mTc ECDG groups were compared (by E.E.K.) with those in the 99mTc ECDG (control) group (three rats).

Scintigrams were obtained by using one of two imaging gamma cameras (Siemens M-camera, Siemens Medical Systems, Hoffman Estates, Ill; or 2020tc, Digirad, San Diego, Calif). Both cameras were equipped with a low-energy, parallel-hole collimator. The field of view with the Digirad camera is 20 x 20 cm with an edge of 1.3 cm. The intrinsic spatial resolution of the Digirad camera is 3 mm, and the matrix is 64 x 64. It is a solid-state gamma camera—that is, it does not have photomultiplier tubes—and is operated in a Windows NT (Microsoft, Redmond, Wash) format. With a preinstalled low-energy, high-spatial-resolution collimator (as is required for use with 99mTc), the system is designed to yield a planar sensitivity of at least 125 counts per minute per microcurie and a spatial resolution of 7.6 mm.

Statistical Analysis
The in vitro percentage of cellular uptake, in vivo percentage of injected dose per gram of wet tissue weight, and tumor-to–nontumorous tissue ratios are presented as means ± standard errors of the means. To compare differences in percentage of cellular uptake between the 99mTc ECDG and 18F FDG groups and the difference between 99mTc ECDG uptake following the addition of D-glucose and that following the addition of L-glucose, the Student t test was used. P < .05 indicated a statistically significant difference. All statistical computations were processed by using a computer software program (Excel; Microsoft).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
Chemistry
The chemical scheme of 99mTc ECDG synthesization is shown in Figure 1. 99mTc ECDG was determined at radio–thin-layer chromatography to have a radiochemical purity of 93.8%–100% (mean, 96.4%) (Fig 2). The yield of radioactivity is dependent on the physical amount of the agent used for radiolabeling. The amount of agent injected for high-performance liquid chromatography was 10 µCi (0.37 MBq)/10 µL/10 µg. The specific radioactivity was calculated to be 0.5 Ci/mmol (18.5 GBq/mmol).



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  		Figure 1. Synthesis of 99mTc ECDG: D-glucosamine is reacted with ethylenedicysteine in the presence of coupling agents, and tin (II) chloride and pertechnetate were added to the solution. MW = molecular weight.

 


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  		Figure 2. ECDG (98.7 mg) was labeled with 100 mCi (3,700 MBq) of NaTcO4 in the presence of stannous chloride and spotted on an instant thin-layer chromatography strip. The eluant used was 1 mol/L of ammonium acetate with methanol (4:1). The radio-thin-layer chromatography data shown indicate 98.5% radiochemical purity. The data shown on the two axes represent counts of 99mTc ECDG and length of the eluant migrated.

 
Hexokinase Assay
A positive hexokinase assay result is characterized by a peak signal in the range of 340–350 nm. The results of hexokinase assay showed that ECDG could be phosphorylated (Fig 3). The findings suggested that hexokinase-catalyzed phosphorylation occurred with ECDG, deoxyglucose, FDG, and glucose. Whether ECDG and deoxyglucose use different glucose transporters needs to be further investigated.



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  		Figure 3. Graph depiction of the results of hexokinase assay performed to determine the phosphorylation of ECDG. ECDG phosphorylation was determined by means of coupling hexokinase activity with the reduction of nicotinamide adenine dinucleotide (NAD) to its reduced form (NADH). The absorption of reduced-form nicotinamide adenine dinucleotide occurs at an ultraviolet wavelength of 340-350 nm; this indicates positive glucose phosphorylation activity (341.5 nm). The absorption of nonreduced nicotinamide adenine dinucleotide occurs at an ultraviolet wavelength of 302.5 nm.

 
In Vitro Cell Culture Studies
There was a marked increase in the uptake of 99mTc ECDG and 18F FDG as a function of time compared with the uptake of 99mTc ethylenedicysteine. 99mTc ECDG had optimal uptake at 4 hours after injection and reached a level of 0.5% administered activity, whereas 18F FDG had a greater than 0.6% uptake at 4 hours after injection (Fig 4). Adding glucose at a concentration of 1–2 mg per well led to a decreased uptake of 99mTc ECDG in lung cancer cells. However, L-glucose had no influence on 99mTc ECDG uptake (Fig 5). These findings suggest that the cellular uptake of 99mTc ECDG is mediated by way of a D-glucose mechanism.



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  		Figure 4. Bar graph illustrates the in vitro cellular uptake of 99mTc ECDG (EC-DG) and 18F FDG. Graph data show that there was a markedly increased uptake of 99mTc ECDG and 18F FDG as a function of time compared with the uptake of 99mTc ethylenedicysteine (EC), the control agent. Asterisks indicate that there was a significant (P < .05, Student pair t test) difference between 99mTc ECDG uptake and 18F FDG uptake during the same interval. Data are reported as means ± standard errors of the mean in the three agent groups. Data points were calculated as percentages of uptake.

 


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  		Figure 5. Bar graph illustrates the in vitro cellular uptake of 99mTc ECDG when glucose (GLU) was administered. Graph data show that there was a markedly decreased uptake of 99mTc ECDG as a function of D-glucose concentration compared with the uptake as a function of L-glucose concentration. Asterisks indicate that there was a significant (P < .05, Student pair t test) difference between the L-glucose- and D-glucose-loaded groups at equal concentrations. Data are reported as means ± standard errors of the mean in the three glucose dose groups. Data points were calculated as percentages of 99mTc ECDG uptake.

 
Effect of D-Glucosamine and FDG Loading on Blood Glucose Level
The blood glucose level increased after the bolus intravenous administration of glucosamine in group 1 and of ECDG in group 2. This increased blood glucose level was suppressed by the administration of insulin in group C (Fig 6). The total number of rodents examined is shown in Table 1.



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  		Figure 6. Data plotted to illustrate the intravenous (i.v.) administration of ECDG (EC-DG) and glucosamine in rats show increasing blood glucose levels as a function of time up to nearly 210 minutes after injection. Intravenous administration of ECDG and insulin caused a marked decrease in blood glucose levels.

 
Tissue Distribution Studies
In group 1, 99mTc ECDG had higher tumor-to–muscle tissue and tumor-to–brain tissue ratios as a function of time, whereas in group 2, 18F FDG had higher tumor-to-blood ratios. There were no significant differences in calculated tumor-to–lung tissue ratios (Tables 2 and 3).


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  		TABLE 2. Biodistribution of 99mTc ECDG in Lung Tumor-Bearing Mice

 

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  		TABLE 3. Biodistribution of 18F FDG in Lung Tumor-Bearing Mice

 
Gamma Scintigraphy Studies
The smallest tumor volume that could be detected by using 99mTc ECDG was 3 mm. There was a significant increase in uptake, as demonstrated by the computer-generated ROI ratios of tumor versus nontumorous uptake as a function of time. The mean ROI ratios of 99mTc ECDG uptake determined immediately, 30 minutes, and 2 hours after radionuclide administration for the tumors versus the corresponding nontumorous regions (ie, in the opposite leg) were 1.70 ± 0.21, 1.58 ± 0.30, and 1.82 ± 0.07, respectively, for the small tumors and 2.36 ± 0.06, 2.41 ± 0.10, and 2.88 ± 0.10, respectively, for the medium-sized tumors. The ranges of ROI counts for the tumor and nontumorous regions were 4,500–16,000 and 2,500–7,000, respectively. The pixel number was determined to be 213. The medium-sized (6-mm) tumors showed higher uptake at each time point. The heart, kidneys, liver, and bladder were visualized (Fig 7). Scintigrams demonstrated that breast tumors in rats could be better visualized with 99mTc ECDG scintigraphy than with 99mTc ethylenedicysteine scintigraphy at similar time points (Fig 8).



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  		Figure 7. Planar scintigrams of 99mTc ECDG (300 µCi [11.1 MBq] per rat administered intravenously) uptake in breast tumor-bearing rats demonstrate that small (3-mm) and medium-sized (med, 6-mm) neoplasms could be imaged up to 2 hours after radionuclide administration. Arrows in the left image point to small and medium-sized tumors. The mean tumor-to-nontumor (opposite leg) ROI ratios determined immediately, 30 minutes, and 2 hours after 99mTc ECDG injection were 1.70 ± 0.21, 1.58 ± 0.30, and 1.82 ± 0.07, respectively, for the small tumors and 2.36 ± 0.06, 2.41 ± 0.10, and 2.88 ± 0.10, respectively, for the medium-sized tumors. Uptake was also observed in the heart (H), liver, kidneys, and bladder.

 


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  		Figure 8. Planar scintigrams of 99mTc ethylenedicysteine (99mTc-EC) and 99mTc ECDG (99mTc-EC-DG) uptake in breast tumor-bearing rats (300 µCi [11.1 MBq] per rat administered intravenously) demonstrate that the tumor could be better imaged at 99mTc ECDG scintigraphy. Arrows point to tumors (T).

 
The rats pretreated with intravenously administered FDG (50 mg per rat) had decreased (40% at ROI analysis) tumor uptake of 99mTc ECDG. However, the rats pretreated with intramuscularly administered insulin (3 units) had increased (60% at ROI analysis) uptake of 99mTc ECDG 0.5–2.0 hours after agent administration. Selected scintigrams obtained 30 minutes after agent administration are shown in Figure 9.



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  		Figure 9. Planar scintigrams of 99mTc ECDG (99mTc-EC-DG) uptake (300 µCi [11.1 MBq] per rat administered intravenously) in breast tumor-bearing rats (three rats per agent group) obtained 30 minutes after injection demonstrate that pretreatment of the rats with FDG (middle image) or insulin (right image) affected the uptake of 99mTc ECDG. Arrow points to the tumor (T).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
 
The findings in the present studies appear to support our initial hypothesis that 99mTc ECDG has the potential to be used as a functional metabolic imaging agent. 99mTc ECDG is positive for phosphorylation at hexokinase assay on the basis of a peak signal in the range of 340–350 nm. This peak represents the ultraviolet absorbency of reduced-form nicotinamide adenine dinucleotide. Nicotinamide adenine dinucleotide, which is included in the hexokinase assay kit, changes to its reduced form during the phosphorylation of glucose in the mitochondria. Thus, we conclude that ECDG, the substrate, is phosphorylated by hexokinase, the enzyme, and that nicotinamide adenine dinucleotide changes to reduced-form nicotinamide adenine dinucleotide, which has a known ultraviolet absorbency that serves as an indicator of a positive assay result.

In addition, the uptake of 99mTc ECDG in lung tumor cell lines is comparable to that of 18F FDG. Both agents accumulate owing to the increased metabolism (ie, increased need for glucose) in proliferating tumor cells. It has been reported that there are at least two mechanisms for the cellular processes of glucosamine (32,33). The first mechanism is similar to the cellular process mechanism of glucose: Glucosamine enters cells by way of a glucose transporter system, and then by way of phosphate and glycolytic pathways, and forms glucosamine-6-phosphate. In the second mechanism, glucosamine enters cells and directly forms glucosamine-6-phosphate. The regulatory products derived from glucosamine-6-phosphate are involved in translocation, transcription, and insulin action cascade. FDG is involved in the glucose phosphate and glycolytic pathways but not in the additional transcriptional pathways. Therefore, 99mTc ECDG may reflect more signaling biosynthetic pathways than 18F FDG.

Overexpression of different glucose transporter types may lead to higher or lower detection specificity for radionuclides. 99mTc ECDG was superior to 18F FDG with regard to tumor-to–brain tissue and tumor-to–muscle tissue ratios, whereas 18F FDG had higher tumor-to-blood ratios. Lower tumor-to-blood ratios of 99mTc ECDG are owing to the higher concentration of this agent in blood compared with the concentration of 18F FDG in blood. This higher concentration may result from the chemical modifications required to synthesize 99mTc ECDG, which can affect uptake kinetics and thus result in an increase in circulation time, flow to the kidneys, and excretion, all of which can also affect tumor uptake. 18F FDG showed higher uptake in the tumor, brain, and heart than did 99mTc ECDG. The lower uptake of 99mTc ECDG by normal brain tissue may have been due to coordination chemistry factors.

99mTc is stabilized by electrons from the nitrogen and sulfur components of ethylenedicysteine; hence, the charge of 99mTc may reduce uptake across the blood-brain barrier and in healthy brain cells. Therefore, brain tumors may be effectively detected with 99mTc ECDG imaging. 18F FDG is a neutral molecule and can cross the blood-brain barrier easily compared with 99mTc ECDG. Thus, FDG PET may not be as effective in the diagnosis of low-grade brain tumors owing to high levels of 18F FDG uptake in normal gray matter. Because of the high tumor-to-background count density ratios generated with 99mTc ECDG imaging, this examination may be more suitable and effective in the detection of low-grade brain tumors than FDG PET.

The principal hypoglycemic hormone is insulin, which is produced in the islets of Langerhans in the pancreas. Insulin, which is secreted in response to an increase in blood glucose level shortly after meals, increases glucose entry into muscle and fat tissues and promotes glycogen synthesis and storage in the liver (34,35). The net result of these actions is a net decrease in blood sugar level. Multiple rats were injected with ECDG or FDG, and, as expected, their blood sugar level increased. The administration of insulin with each agent led to a dramatic and similar decrease in blood sugar level in each animal, demonstrating that the two agents have similar uptake mechanisms. The response to insulin may be an important parameter for physicians to consider for patients with diabetes who are administered 99mTc ECDG for imaging. Insulin will lower their blood sugar levels after the administration of 99mTc ECDG and thus prevent hyperglycemia, and it may allow a larger population of patients to participate in 99mTc ECDG imaging studies.

The feasibility of performing imaging with 99mTc ECDG was evaluated in mammary tumor–bearing rats with tumors in the hind leg. First, the agent was compared with 99mTc ethylenedicysteine and enabled good visualization of the tumors at 2 and 4 hours after injection. 99mTc ethylenedicysteine is a blood flow agent that has no detection specificity for any tissue or organ. 99mTc ethylenedicysteine was delivered to the liver and kidneys owing to high levels of blood flow through these organs; the agent remained in these regions mainly owing to interactions between ethylenedicysteine, acetylcysteine (in the kidneys), and glutathione (in the liver and kidneys), with the result of trapping of the 99mTc ethylenedicysteine molecule. Having established the effectiveness of 99mTc ECDG in targeting tumor cells, we performed another study to evaluate the usefulness of this agent in the detection of small (3-mm) and medium-sized (6-mm) tumors. The agent effectively enabled the detection of each size of tumor up to 2 hours after injection while remaining stable, as evidenced by the low uptake in the thyroid gland, which suggested in vivo stability. Finally, the rats pretreated with FDG and injected with 99mTc ECDG had decreased tumor uptake compared with the rats that were not pretreated. This finding indicates that 99mTc ECDG has an uptake mechanism similar to that of FDG—that is, the two agents have the same transporter types—and that the uptake of 99mTc ECDG is inhibited by the presence of FDG (competitive inhibition). As expected, the effects of insulin, mimicking those of normal glucose, resulted in increased 99mTc ECDG tumor uptake.

Practical application: There are similarities between the uptake of 99mTc ECDG and the uptake of 18F FDG in tumors, and our findings support the potential use of 99mTc ECDG as a functional metabolic imaging agent. In addition, ethylenedicysteine can be labeled with 99mTc easily, efficiently, and with high radiochemical purity, stability, and cost effectiveness. With the complex mechanisms expressed in tumor tissue growth, the described 99mTc ethylenedicysteine and drug conjugate technique allows mechanism-specific imaging of cellular targets with use of 99mTc ECDG. This examination has the potential to improve the diagnosis before and prognosis, planning, and monitoring of cancer treatment.


   ACKNOWLEDGMENTS
 
The authors thank Eloise Daigle for her secretarial support.


   FOOTNOTES
 
Abbreviations: ECDG = ethylenedicysteine– deoxyglucose, FDG = fluorodeoxyglucose, ROI = region of interest

Author contributions: Guarantor of integrity of entire study, D.J.Y.; study concepts, E.E.K., N.R.S.; study design, D.J.Y., D.F.Y., E.E.K.; literature research, J.J.W., J.L.B.; clinical studies, E.E.K., C.G.K., D.A.P.; experimental studies, A.A., D.F.Y.; data acquisition, A.A., C.S.O.; data analysis/interpretation, A.A., J.L.B.; statistical analysis, A.A.; manuscript preparation, D.J.Y.; manuscript definition of intellectual content, D.J.Y., E.E.K., D.F.Y.; manuscript editing, D.A.P., E.E.K., C.G.K.; manuscript revision/review, E.E.K., D.J.Y.; manuscript final version approval, D.J.Y.


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 MATERIALS AND METHODS
RESULTS
DISCUSSION
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