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The purposes of this study were to develop an efficient method of labeling D-glucosamine hydrochloride with gallium 68 (68Ga) and investigate the imaging properties of the resulting radiotracer in a human tumor xenograft model using micro-positron emission tomography (μPET). The precursor compound 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-2-deoxy-D-glucosamine (DOTA-DG) was synthesized from D-glucosamine hydrochloride and 2-(4-isothiocyanatobenzyl)-DOTA. Radiolabeling of DOTA-DG with 68Ga was achieved in 10 minutes using microwave heating. The labeling efficiency a nd radiochemical purity after purification of 68Ga-DOTA-DG were ~85% and greater than 98%, respectively. In A431 cells, the percentages of 68Ga-DOTA-DG and 18F-FDG uptakes after 60 min incubation were 15.7% and 16.2%, respectively. In vivo, the mean ± standard deviation of 68Ga-DOTADG uptake values in A431 tumors were 2.38±0.30, 0.75±0.13, and 0.39±0.04 percent of the injected dose per gram of tissue at 10, 30, and 60 minutes after intravenous injection, respectively. μPET imaging of A431-bearing mice clearly delineated tumors at 60 minutes after injection of 68Ga-DOTA-DG at a dose of 3.7 MBq. 68Ga-DOTA-DG displayed significantly higher tumor-to-heart, tumor-to-brain, and tumor-to-muscle ratios than 18F-FDG did. Further studies are needed to identify the mechanism of tumor uptake of this new glucosamine-based PET imaging tracer.
The positron-emitting radioisotope gallium 68 (68Ga; t 1/2 = 68 minutes) is of great interest to practitioners of positron emission tomography (PET). This generator-produced nuclide does not require an on-site cyclotron for radioisotope production. 68Ga has excellent physical properties relevant to PET, including decays by 89% through positron emission and 3.2% through gamma emission . A number of 68Ga-labeled peptides have been investigatror for in vivo PET imaging of tumors [2,3]. 18F-fluorodeoxyglucose (FDG) is routinely used in the clinic as a biomarker of metabolic activity for the detection and staging of cancer and monitoring of treatment response. 18F-FDG is transported into tumor cells by the glucose transporters (GLUTs) and is a substrate of hexokinases. D-glucosamine (DG) is an attractive scaffold as a glucosyl ligand. Studies have shown that a derivative of DG with a bulky moiety attached to its amino group is the substrate of GLUTs and hexokinases [4,5].
Previously, the synthesis of 68Ga-labeled glucosamine through 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) radiometal chelator have been reported by us and others in abstract forms [6,7]. Tworowska et al.  synthesized 68Ga-DOTA-glucosamine by heating to 95 °C using conventional method for 10-20 min. To improve the radiolabeling efficiency, we used microwave to assist the labeling process. Microwave heating is used routinely in the synthesis of organic compounds . This heating increases the rate of reaction without compromising the stability of reagents or the safety of labeling procedures [9,10]. Herein we report on our use of microwave heating-assisted labeling of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-DG with 68Ga for tumor imaging. We found that the resulting radiotracer, 68Ga-DOTA-DG, could clearly delineate tumor xenografts in nude mice with high tumor-to-nontumor ratios, making it a potentially useful radiotracer in PET imaging.
Dimethylformamide, sodium acetate, D-glucosamine hydrochloride, and N-methylmorpholine were purchased from Sigma-Aldrich (St. Louis, MO). 2-(p-Isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid (p-SCN-Bn-DOTA) was purchased from Macrocyclics (Dallas, TX). Other commercially available chemicals were purchased from VWR International (San Diego, CA). All reagents were used as received. 18F-FDG was obtained from the Department of Nuclear Medicine at The University of Texas MD Anderson Cancer Center.
DG was reacted with p-SCN-Bn-DOTA in an aqueous solution at pH 7 for 12 hours at room temperature to produce DOTA-DG linked via a thiourea bond (Figure 1). The progress of the reaction was monitored using high-performance liquid chromatography (HPLC). DOTA-DG was analyzed via electrospray ionization mass spectrometry using an Agilent LC/MSD TOF mass spectrometer (Agilent Technologies, Santa Clara, CA) equipped with a Vydac C-18 column (4.6×250.0 mm, 7-μm particle size, 300-Å pore size (Grace, Deerfield, IL). The HPLC eluting conditions were as follows: solvent: A, 0.1% trifluoroacetic acid (TFA) in water; B, 0.1% TFA in acetonitrile; gradient: B, 0-10% over 0-10 minutes; B,10-50% over 10-12 minutes; B, 50-80% over 12-20 minutes; B, 80% to 10% over 20-21 minutes. The flow rate was 1 mL/minute.
68GaCl3 in 0.1 N hydrochloric acid (370 MBq/0.3 mL) was obtained from 68Ge/68Ga generator (Eckert & Ziegler, Berlin, Germany). Two methods were used for labeling of DOTA-DG with 68Ga. In the conventional method, 370 MBq of 68GaCl3 in 0.3 mL of a 1-M aqueous solution of sodium acetate (pH 4) was added to a solution of DOTA-DG in distilled water (100 μg in 0.1 mL). The mixture was allowed to react at 90°C for 20 minutes in a water bath and then cooled to room temperature over 30 minutes. In the microwave heating-assisted method, the mixture of 370 MBq of 68GaCl3 in 0.3 mL of a 1-M sodium acetate solution and 100 μg of DOTADG in 0.1 mL of distilled water was heated in a microwave reaction apparatus (Discover; CEM Corporation, Matthews, NC) at a power setting of 25 W for 5 minutes and 50 W for an additional 5 minutes. The reaction mixture was then kept at 40°C for 5 minutes. DOTA was labeled with 68Ga similarly to DOTA-DG, and the resulting radiotracer, 68Ga-DOTA, was used as a control in biodistribution study.
Radiolabeled compounds were analyzed using HPLC. The specific activity of 68Ga-DOTA-DG was determined with a radio-HPLC chromatogram by dividing the integrated peak radioactivity of the radiotracer by its physical quantity derived from the corresponding ultraviolet absorbance and a calibration curve of known quantities of the unlabeled compound. The labeling yields were determined using instant thin-layer chromatography (ITLC) (German Science, Ann Arbor, MI) developed with a solution of 0.1 M citric acid in saline. The strips were scanned using an ITLC scanner (AR-2000; Bioscan, Washington, DC).
A431 human epithelial carcinoma cells were obtained from the American Type Culture Collection (Manassas, VA). Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2 in Dulbecco’s modified Eagle’s medium and Ham’s F12 nutrient mixture containing 10% fetal bovine serum (Gibco, Grand Island, NY).
Cell uptake assays were performed after seeding 1.2×106 A431 cells/mL/well in 24-multiwell culture plates. When the cells were about 80% confluent, each well was injected with 74 KBq of 68Ga-DOTA-DG or 18F-FDG in 1 mL of culture media (2 μg/mL for both radiotracers). A blocking study was performed with the addition of both radiotracers with 1 mg of cold deoxy-Dglucose to the wells (100 μL/well). After 60 minutes of incubation at 37°C, the culture media were removed, and the cells were washed twice with ice-cold Hank’s balanced salt solution (pH 7.3). The cells were then dissolved in 0.5% sodium dodecyl sulfate (0.5 mL/well). The radioactivity in the cells was measured using a gamma counter (Packard, Downers Grove, IL). The cell uptake of the radiotracers was calculated using the formula %uptake = (radioactivity of cells/total radioactivity) × 100%. This study was performed in triplicate.
The mice were kept under specific pathogen-free conditions and were handled and maintained according to Institutional Animal Care and Use Committee guidelines. A431 cells were inoculated subcutaneously into the right thighs of nude mice (20-25 g; Harlan Sprague Dawley, Indianapolis, IN) by injecting 1×106 viable tumor cells in a suspension of phosphate-buffered saline. When the resulting tumors grew to a diameter of 6-8 mm, the mice were allocated to three groups of three mice each. They were then injected intravenously with 68Ga-DOTA-DG or 18F-FDG (3.7 MBq/mouse). The animals were killed at 10, 30, and 60 minutes after injection. Blood, heart, liver, spleen, kidney, lung, stomach, intestine, muscle, bone, brain, and tumor tissues were removed, weighed, and counted for radioactivity using a gamma counter. Uptake of each radiotracer in various tissues was calculated as the percentage of the injected dose per gram of tissue (%ID/g).
A431 tumor cells were inoculated into nude mice as described above. When the resulting tumors grew to 6-8 mm in diameter, the mice were injected intravenously with 68Ga-DOTA-DG (20 μg, 14.8 MBq/mouse, 0.2 mL). The mice were then placed in the prone position for micro-PET (μPET) imaging. Prior to imaging, the mice were anesthetized with 2% isoflurane gas (Iso-Thesia, Rockville Centre, NY) in oxygen. During imaging, anesthesia in the mice was maintained with 0.5-1.5% isoflurane. μPET images were acquired 30-80 minutes after radiotracer injection using an R4 μPET scanner (Concorde Microsystems, Knoxville, TN).
Cell uptake and biodistribution data were analyzed using two-tailed, unpaired Student t-tests, with p values less than 0.05 considered to be statistically significant. The in vitro percentage of radiotracer uptake, in vivo percentage of injected radiotracer dose per gram of tissue, and tumor-to-nontumor ratios are presented as the mean ± standard deviation. All statistical computations were performed using the Excel software program (Microsoft Corporation, Redmond, WA).
DOTA-DG was synthesized via nucleophilic addition of DG to p-SCN-Bn-DOTA (Figure 1). The resulting product was purified via flash column chromatography. Electrospray ionization mass spectrometry revealed a mass/charge ratio (m/z) of 729.2082 for [M-H]-, which agrees with the calculated value for C30H45N6O13S- of 729.2771. HPLC analysis demonstrated a retention time of 7.61 minutes with greater than 90% DOTA-DG purity (Figure 2).
68Ga-DOTA-DG was synthesized using two methods: conventional and microwave-assisted heating. The radiochemical labeling efficiency of 68Ga-DOTA-DG with the microwave heating technique was 85%. After purification of 68Ga-DOTA-DG using a semipreparative HPLC column, the radiochemical purity was greater than 98% (Rt = 7.01 minutes). Radio-HPLC readily distinguished 68Ga-DOTA-DG from 68GaCl3 (Rt = 3.15 minutes) and 68Ga-DOTA (Rt = 2.92 minutes) (Figure 3). In comparison, the radiochemical labeling efficiency for 68Ga-DOTA-DG using the conventional method was 60%. The specific activity of 68Ga-DOTA-DG using the microwave heating method was 790 Ci/mmol (2.9 × 1013 Bq/mmol).
In vitro, 68Ga-DOTA-DG and 18F-FDG had similar uptake levels in A431 cells after 60 minutes of incubation (Figure 4). The percentages of 68Ga-DOTA-DG and 18F-FDG uptake were 15.7% and 16.2%, respectively. The presence of an excessive amount of cold deoxy-D-glucose (1 mg/mL water) significantly blocked the uptake of 18F-FDG in these cells (p<0.001). However, deoxy-D-glucose could not block 68Ga-DOTADG. To the contrary, the presence of cold deoxy-D-glucose increased the uptake of 68Ga-DOTA-DG in A431 cells by 121% (p=0.03).
The biodistributions of 68Ga-DOTA-DG at 10, 30, and 60 minutes after radiotracer injection in mice bearing A431 tumors are presented in Table 1. A comparison of the biodistributions of 68Ga-DOTA, 68Ga-DOTA-DG, and 18F-FDG at 60 minutes postinjeciton are presented in Table 2. The tumor-to-nontumor ratios for 68Ga-DOTA-DG and 18F-FDG are shown in Figure 5.
The 68Ga-DOTA-DG blood activity at 10 min postinjection was 5.24 %ID/g, which is similar to that of the reported blood level of 5.4 %ID/g for 18F-FDG . The initial 68Ga-DOTA-DG tumor uptake value at 10 minutes after injection was 2.38%ID/g. At 60 minutes after injection, the tumor uptake values for 68Ga-DOTA, 68Ga-DOTA-DG, and 18F-FDG were 0.20%ID/g, 0.39%ID/g, and 4.26%ID/g, respectively. The blood activity levels at 60 min postinjection were similar for both 68Ga-DOTA-DG (0.40 %ID/g) and 18F-FDG (0.54 %ID/g) (Table 2). Thus, 18F-FDG had significantly higher tumor-to-nontumor ratios than 68Ga-DOTA-DG did at 60 minutes after injection in blood (p=0.05), liver (p=0.03), and kidney (p=0.02). On the other hand, 68Ga-DOTA-DG had significantly higher tumor-to-nontumor ratios than 18F-FDG did in muscle (p=0.05), brain (p=0.02), and heart (p=0.02) (Figure 5).
μPET images of mice with A431 tumors obtained at different times after 68Ga-DOTA-DG injection are shown in Figure 6. We observed relatively high activity of 68Ga-DOTA-DG in the kidney and bladder 30-60 minutes after injection. The activity of 68Ga-DOTA-DG in the kidney gradually cleared, and by 80 minutes after injection, 68Ga-DOTA-DG was largely cleared from the body via the renal system. μPET imaging clearly showed uptake of 68Ga-DOTA-DG in the tumors at all time points.
In this study, we showed that DOTA-DG could be labeled with 68Ga with high efficiency with the assistance of microwave. The observation that the cellular uptake of 68Ga-DOTA-DG could not be blocked by a large excess of D-glucose suggests that 68Ga-DOTA-DG is taken up by tumors cells via biological processes independent of GLUTs (Figure 4). At present, the structural features that govern the behavior of derivatives of DG are not clear. Although previous studies demonstrated that DG and its fluorescent analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxyglucose are substrates of GLUTs [4,5,12], the uptake of other derivatives of DG in tumor cells could not be blocked by D-glucose or DG. For example, several 99mTc- and nearinfrared fluorescent dye-labeled DG compounds have similar characteristics as 68Ga-DOTA-DG with respect to insensitivity to glucose concentration on their tumor uptake in vivo [13-15].
Several authors have reported the synthesis and in vivo imaging with gamma emitter-labeled DG. Bayly et al.  reported the successful synthesis of DG labeled with the tricarbonyls of 99mTc (I), but the resulting radiotracer was not very stable in the presence of cysteine and histidine. Yang et al.  demonstrated that 99mTc-labeled DG through an ethylenedicysteine chelator accumulated in murine tumors. Chen et al.  developed a one-step 99mTc-labeled diethylenetriaminepentaacetate-DG kit and showed accumulation of 99mTc-labeled diethylenetriaminepentaacetate-DG in MCF-7 human mammary tumors in nude rats. These studies did not attempt to determine whether these glucose analogs are actually involved in key steps in glucose metabolism. Further work is needed to delineate the mechanisms of cellular uptake of 68Ga-DOTA-DG and other DG-based radiotracers.
μPET images acquired with 68Ga-DOTA-DG clearly delineated A431 tumor grown in nude mice. With the current available data, it is not possible to ascertain whether the tumor uptake of 68Ga-DOTA-DG is specific or nonspecific. Although DOTA is used widely for 68Ga-labelling, DOTA is not an optimal chelator for 68Ga. Other chelators such as 1,4,7-triazacyclononane-triacetic acid (NOTA) and triazacyclononane-phosphinate (TRAP) chelators have been shown to possess superior 68Ga binding ability and yield higher specific activity . Thus, future work should be also be directed at synthesizing and testing 68Ga-NOTA-glucosamine and 68Ga-TRAPglucosamine conjugates.
In conclusion, we successfully synthesized 68Ga-labeled DOTA-DG as a PET radiotracer. Microwave heating-assisted radiosynthesis of 68Ga-DOTA-DG resulted in high labeling efficiency and short labeling times. μPET with 68Ga-DOTA-DG demonstrated high tumor-to-nontumor ratios for muscle, brain, and heart in human tumor xenograft models. We also observed a significant difference between the biodistribution of 68Ga-DOTA-DG and 18F-FDG in the liver and kidney. Future work are needed to elucidate the biological processes responsible for tumor uptake of 68Ga-DOTA-DG.
The authors thank Donald R. Norwood for editing the manuscript. This work was supported in part by the John S. Dunn Foundation and by grant #81071198 and grant #81172082 from the National Natural Science Foundation of China. The small animal imaging study is supported by the MD Anderson Cancer Center Support Grant CA016672.