Synthesis 99mTc-DTPA-deoxy-D-glucose

(99mTc-DTPA-DG) as tumor imaging

Cite as: AIP Conference Proceedings 2381, 020038 (2021); https://doi.org/10.1063/5.0067021
Published Online: 11 November 2021

Eva Maria W., Nunik Utari N., Maula Eka S., et al.

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© 2021 Author(s).
 Synthesis 99mTc-DTPA-Deoxy-D-Glucose (99mTc-DTPA-
DG) as Tumor Imaging
Eva Maria W1,a), Nunik Utari N2), Maula Eka S1), Witri Nuraeni1), and
Teguh Hafiz AW 2)
1
Center for Applied Nuclear Science and Technology-BATAN, Jl. Tamansari No. 71 Bandung, Indonesia
2.
Pharmacy Study Program Faculty of Pharmacy - Universitas Jendral Achmad Yani
Jl Terusan Jendral Sudirman Cimahi, Indonesia
a)
Corresponding author: evamaria@batan.go.id.

Abstract. Cancer is the uncontrolled growth of new cells beyond the normal limits. The process by which cancers invade
and spread to the other organs is called metastasis, this condition became the leading cause of death. One of detection
methods that are currently used to detect tumors and metastatic tissues is conducted with radiopharmaceutical of [ 18F]
fluoro-2-deoxy-2-D-glucose ([18F] FDG). This method is quite accurate, but is relatively expensive because it needs PET
/ CT camera that is still limited in Indonesia. Therefore, this study was conducted to develop other imaging methods using
SPECT-CT camera that is cheaper and easy to reach especially for developing countries. Labelling 2-deoxy-D-glucose
with technetium-99m (99mTc) indirectly using diethylenetriaminepentaacetic acid (DTPA) as a co-ligand / bifunctional
agent has been carried out. The determination of the optimum conditions for the labelling of 99mTc-DTPA-DG were done
by varying the amount of reducing agent (SnCl2.2H2O), ligand (2-deoxy-D-glucose), pH, and incubation time.
Radiochemical purity of 99mTc-DTPA-DG was determined by ascending paper chromatography and electrophoresis paper.
The results showed that the labelling optimum conditions of 99mTc-DTPA-DG was achieved by the number of ligand 2-
deoxy-D-glucose as much as 2 mg, co-ligand DTPA 750 µg, 50 µg SnCl2.2H2O, the reaction takes place at pH 6 with an
incubation time of 30 min at room temperature. 99mTc-DTPA-DG has a radiochemical purity of 93.16 ± 1.31% and the
electrophoresis results showed that there is a difference between the peaks of 99mTc-DTPA-DG with other impurities
including 99mTc-DTPA.

INTRODUCTION
Cancer is the leading cause of death in both developing and developed countries. The incidence of cancer increases
with population growth and aging, as well as the increasing prevalence of risk factors such as smoking, being
overweight, poor diet, lack of physical activity, and changing reproductive patterns associated with urbanization and
economic development. According to World Health Organization (WHO), cancer is a general term for a large group
of diseases that can affect any part of the body. Other terms used are malignant tumors and neoplasms. One of the
defining features of cancer is the abnormal growth of new cells that grow beyond normal limits so that they can invade
and spread to other organs [1]. This process is called metastasis, which is the leading cause of cancer death. Based on
GLOBOCAN data, approximately 14.1 million new cancer cases and 8.2 million deaths occurred in 2012 worldwide
[2]. Early detection, accurate diagnosis and prompt and precise treatment can help reduce the suffering that is felt and
increase the life expectancy of sufferers.
Nuclear imaging using either Single Photon Emission Computed Tomography (SPECT) or Positron Emission
Tomography (PET) is very important molecular imaging because it is very sensitive to the amount of radioactive
substances used as tracers [3]. Both of these tools have advantages and disadvantages. PET imaging provides more
sensitive results, has better spatial resolution and can determine the quantity of the target. Whereas SPECT is less
expensive and is considered a more practical approach in nuclear medicine for routine diagnostic use. Both modalities

Proceedings of International Conference on Nuclear Science, Technology, and Application 2020 (ICONSTA 2020)
AIP Conf. Proc. 2381, 020038-1–020038-8; https://doi.org/10.1063/5.0067021
Published by AIP Publishing. 978-0-7354-4153-8/$30.00

020038-1
are widely used to characterize and measure biological characteristics at the cellular and molecular levels of living
things[4].
In imaging intended for cancer diagnosis, metabolism is a process that plays an important role. The metabolism
that occurs in cancer cells is faster than normal cells, so that the energy required is also greater. Glucose is an important
molecule needed in metabolic processes because glucose is a source of energy for living things. Therefore, the
absorption of glucose by cancer cells will be greater than normal cells so that glucose can be used as a candidate as a
compound for tumor detection [4]. Many studies have successfully used glucose or glucose derivatives as
radiopharmaceuticals for tumor detection. 2-Deoxy-D-glucose (DG) is a synthetic analogue of glucose in which the
hydroxyl group on the second position carbon is replaced by hydrogen. DG inhibits hexokinase and glucose phosphate
isomerase thereby blocking glycolysis. Therefore, DG is expected to cause a decrease in ATP and glucose derivatives
required for protein glycosylation [5], [6]. In many studies, disruption of glycolysis or energy depletion (cells getting
low or no glucose intake) has been simulated in vivo in animals and in in-vitro culture by adding DG in the medium.
DG alone or in combination with other tumor therapies effectively blocks tumor cell growth in animal models and in
various human tumor cells [6]. Cancer cells treated with 2-deoxiglucose exhibited a stress response caused by
depletion of intracellular energy. The stress response results in increased levels of glucose transporter expression and
increased glucose uptake which allows more 2-deoxyglucose to enter the cells. The high concentration of intracellular
2-deoksiglucose causes hexokinase and hexose phosphate isomerase to be inhibited so that stored energy such as ATP
is used up more quickly so that the cell will activate the cell death pathway [7].
18
F-2-fluoro-2-deoxy-D-glucose ([18F] FDG), is an analogue of glucose which can enter the cell membrane through
the transpot system such as glucose. [18F] FDG will then undergo a phosphorylation reaction by hexokinase, but this
metabolite does not undergo further metabolism and remains trapped in cells [8]. Therefore [18F] FDG is used as a
radiopharmaceutical for tumor detection using PET. Although metabolic imaging of tumors with [ 18F] FDG has been
widely used, its clinical use is limited in practice. This is due to several factors, such as difficult access to PET services
due to limited availability and expensive imaging costs. In addition, the setup of the [18F] FDG had to be done quickly
because the half-life of the F-18 was only 109 minutes. Thus, it is necessary to develop other imaging agents that are
cheaper and easier to reach, especially for developing countries, where the use of SPECT is still dominant.
Technetium-99m is an ideal radioisotope (t½1 = 6.01 hours, energy = 140 keV) for applications with SPECT and can
be obtained using a 99Mo/99mTc generator which is cheaper than F-18 radioisotope.
In this study, the labelling of the 2-deoxy-D-glucose compound (Figure 1) using the technetium-99m radioisotope.
As in research that has been conducted by several previous researchers [9], the labelled was carried out indirectly
using diethylene triamine pentaacetate (DTPA) as a bifunctional agent. The labelled compound 99mTc-DTPA-DG is
expected to be used as a tumor imaging such as [18F] FDG but its application uses the SPECT facility.

FIGURE 1. 2-Deoxy-D-glucose structure

MATERIALS AND METHOD

Materials
The materials used in this study include: 2-deoxy-D-glucose (Sigma Aldrich), tin (II) chloride / SnCl2 (Sigma-
Aldrich), diethyl triamine pentaacetate / DTPA (Fluka), sterile aquabidest (IPHA Laboratories), physiological NaCl
solution (IPHA Laboratories), HCl (E.Merck), NaOH (E.Merck), acetone (E. Merck), 99Mo / 99mTc generator (Ansto)
and other reagents produced by E. Merck with pro-analysis purity. The supporting materials used are pH indicator

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(E.Merck), Instant Thin Layer Chromatography - Silica Gel (ITLC-SG) (Agilent), Whatman 3MM (E.Merck) and
Whatman 1 (E.Merck). The equipment used in this research includes: Dose Calibrator (Victoreen), Vortex Mixer,
Single Channel Analyzer (Ortec), TLC scanner (BIOSCAN), a set of chromatography tools, a set of electrophoresis
tools and glassware.

Preparation of Sn-DTPA, 2-Deoxy-D-glucose, and Na99mTcO4 Solutions

Sn-DTPA solution was prepared by adding 5 mg of SnCl2.2H2O to the vial containing 75 mg of DTPA in 5 mL of
aquabidest. The vial containing Sn-DTPA solution was then vacuumed and stored at room temperature. 2-deoxy-D-
glucose solution is prepared by dissolving 2-deoxy-D-glucose in aquabidest at a concentration of 10 mg/mL.
Na99mTcO4 solution was eluted from a 99Mo /99mTc generator.

Optimization of pH Reaction
To the six vials, each containing 200 μL of 2-deoxy-D-glucose (10 mg / mL) solution was added with 60 μL of
Sn-DTPA solution. The acidity level of the solution in each vial was varied at pH 3, 4, 5, 6, 7, and 8, by adding
dropwise 0.1 N HCl solution or 0.1 N NaOH solution. The volume in each vial was equalized by the addition of
aquabidest sterile. To each vial was added 500 μL of Na99mTcO4 with an activity of ± 1 mCi and that the final volume
was 1 mL. The final pH condition was checked with pH indicator paper. Furthermore, the solution was incubated for
30 minutes at room temperature. The labelled efficiency was determined based on the radiochemical purity using
chromatographic method. The same procedure is carried out for blanks.

Optimization of the Reducing Agent SnCl2.2H2O

Into four vials, each containing 200 μL of 2-deoxy-D-glucose solution (10 mg / mL) were added with a Sn-DTPA
solution with a volume of 40, 50, 60, and 70 μL with varying amounts of SnCl2.2H2O 40, 50, 60, and 70 μg,
respectively. The volume in each vial was equalized by the addition of a sterile aquabidest. The solution in each vial
was adjusted to pH 6 with the addition of 0.1N HCl solution. After that, 500 μL of Na99mTcO4 was added with an
activity of ± 1 mCi so that the final volume of the solution was 1 mL and the final pH was 7. Then the solution was
incubated for 30 minutes at room temperature. The labelled efficiency is determined based on the radiochemical purity
using chromatographic method. The same procedure is carried out for blanks.

Optimization of 2-deoxy-D-glucose Ligand Levels

Into four vials, each containing 100, 200, 300, and 400 μL of 2-deoxy-D-glucose solution, which each contain 2-
deoxy-D-glucose 1, 2, 3, and 4 mg, then each vial was added with aquabidest to 400 μL. Furthermore, each vial was
added with a solution of Sn-DTPA with a volume of 50 μL. The volume in each vial was equalized by the addition of
a sterile aquabidest. The solution in each vial was adjusted to pH 6 by adding dropwise 0.1N HCl solution. After that,
500 μL of Na99mTcO4 was added with an activity of ± 1 mCi so that the final volume of the preparation was 1 mL and
the final pH was 7. Then the solution was incubated for 30 minutes at room temperature. The labelled efficiency is
determined based on the radiochemical purity using chromatographic method. The same procedure is carried out for
blanks.

Optimization of Incubation Time

To a vial containing 200 μL of 2-deoxy-D-glucose solution (10 mg / mL) was added a solution of Sn-DTPA
with a volume of 50 μL. The volume in each vial was equalized by the addition of a sterile aquabidest. The solution
in each vial was adjusted to pH 6 by adding dropwise 0.1N HCl solution. After that, 500 μL of Na99mTcO4 was added

020038-3
with an activity of ± 1 mCi so that the final volume of the preparation was 1 mL of final pH 7. Then the solution was
divided into 2 parts, one part was incubated at room temperature and the other part was incubated in the refrigerator
(-5 ° C) . The variations of the incubation times were 0, 15, 30, 45, and 60 minutes. When the incubation time is
reached, the labelled efficiency is measured which is determined based on the radiochemical purity using
chromatographic method. The same procedure is carried out for blanks.

Determination of Radiochemical Purity of 99mTc-DTPA-Deoxy-D-glucose

The radiochemical purity (RCP) determination of 99mTc-DTPA-deoxy-D-glucose was carried out by a combination
of ascending paper chromatography using Whatman 3MM paper and ITLC-SG. Each paper and plate with a size of
1x10 cm is marked on each centimeter starting with the numbers -1, 0, 1, 2, 3, 4, 5, 6, 7, and 8. Before using the
chromatography paper, it is heated for a few minutes in the oven to remove water content, especially in ITLC-SG.
99m
Tc-DTPA-deoxy-D-glucose is spotted on chromatogram paper, at zero point. On another paper, Na99mTcO4 and
99m
Tc-DTPA are also spotted as blanks. After that the paper is hung on the hook and eluted with eluent. The 3MM
Whatman paper stationary phase was eluted using the dry acetone mobile phase to identify the radiochemical
impurities in the form Na99mTcO4 and the ITLC-SG stationary phase was eluted with the mobile phase of 0.9 % NaCl
solution, to identify reduced Na99mTcO4 (99mTcO2) radiochemical impurities. After the eluent reaches 8 point, the
elution process was stopped and chromatography paper dried. After the paper is dry, the chromatogram paper is cut
every centimeter and then the radioactivity was counted using single channel analyzer (SCA). The count can also be
done using a TLC scanner, but the chromatogram paper is not cut, the chromatogram is placed on an aluminum plate
and scanning is done automatically for each chromatogram. The same procedure is carried out for blanks.

RESULTS AND DISCUSSION

According to coordination chemistry, 2-Deoxy-D-glucose has a structure similar to glucose with a functional
group that is rich in weak electron donors. Therefore, in this study the labelled of 2-deoxy-D-glucose compound with
technetium-99m radionuclide was carried out by the indirect method using DTPA as a bifunctional agent. The
selection of DTPA (Figure 2) as a bifunctional agent is based on the chemical properties of technetium-99m which
readily reacts with hydrophilic compounds and has electron donor atoms such as O, N, and S. DTPA is a chemical
compound that is rich in electron donor groups so that with can easily bind with technetium-99m to form a 99mTc-
DTPA complex (Figure 3)[10]. As previously reported, based on the literature review and the determination of the
electronegativity of the 2-deoxy-D-glucose compound, the labelled compound 99mTc-DTPA-DG is predicted to have
a complex compound structure as shown in Figure 3 [11].
Optimization of a labelled condition is a process carried out to obtain a formulation which when a radioisotope is
added to it, a high labelling efficiency and radiochemical purity will be obtained. In this optimization process, a
number of variables that affect the labelling process are carried out, such as reaction pH, reducing agent level, ligand
content and time and conditions reaction. In this study, the first variable to be varied was the pH level, the results of
the pH variation are presented in Figure 4. From Figure 4 it can be seen that the optimum pH is at pH 6 with
radiochemical purity 94.18 ± 1.44%.

FIGURE 2. Chemical structure: a. Diethyl Triamine Pentaacetate (DTPA) b. 99mTc-DTPA

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 FIGURE 3. Predicted structure of the 99mTc-DTPA-DG complex

FIGURE 4. pH optimization on the 99mTc-DTPA-DG labelling

After the optimum pH is obtained, the research is continued on the next variable, that is reducing levels. The
reducing agent is an indispensable component in the process of labelling a compound with technetium-99m
(Na99mTcO4) radioisotope. 99mTcO4- is a chemical compound that is not reactive and cannot be used directly to label
a compound [12]. Therefore we need a reducing agent which can reduce the technetium-99m oxidation number from
Tc (VII) to a lower one. The oxidation number suitable for technetium-99m reacting with DTPA is +4 [Tc (IV)] [12].
In this study, the reducing agent used was SnCl2.2H2O, and from the results shown in Figure 5 it can be seen that the
optimum reducing agent level is 50 µg.
Another variable that determines the success of the marking is the ligand (2-deoxy-D-glucose). Although several
studies on the preparation of marked compounds did not vary the ligand and the ligand content was considered a fixed
variable, in this study there were still variations in the ligand content. This is done to see the effect of ligand levels on
radiochemical purity (RCP) of 99mTc-DTPA-DG. As shown in Figure 6, variations in ligand levels do not an effect on
RCP of 99mTc-DTPA-DG and the optimum reaction at 2 mg ligand levels.

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 FIGURE 5. SnCl2 levels optimization on the 99mTc-DTPA-DG labelling

FIGURE 6. 2-deoxy-D-glucose levels optimization on the 99mTc-DTPA-DG labelling

The last variation is incubation time. The results of the optimization test for the incubation time at room
temperature are shown in Figure 7 which shows that the optimum incubation time is 30 minutes after the addition of
technetium-99m. In addition, incubation was also carried out in the refrigerator (4oC), in this condition the optimum
incubation time was 45 minutes after the addition of technetium-99m (Figure 8). This shows that at low temperatures
the reaction will run slowly because at low temperatures the kinetic energy of the reacting molecules will decrease.
The high variation data in iteration at variations in incubation time (4oC) causes a large standard deviation so that the
data at the adjacent test times when calculating the standard deviation value will coincide. This shows that the
difference in the incubation time of 15 minutes gives a result that is not too different because the reaction is slow. But,
from the average value, the incubation time of 45 minutes is the optimum. And based on the variation of the
incubation time data, the incubation should be done at room temperature for 30 minutes and after use the labelled
compound is stored in the refrigerator to maintain the RCP of the labelled compound.
As explained earlier, the labelling method used in this study is an indirect method using DTPA as the bifunctional
agent. So that the reaction that occurs is as follows:

Ca-DTPA + Sn(2+)Cl2 CaCl2 + Sn(2+)-DTPA …………………………………………………..............................1

Sn(2 +)-DTPA + 2-deoxy-D-glucose (DG) Sn(2+) - DTPA-DG + Sn(2+)-DTPA ......................................................2

Sn(2 +) -DTPA-DG + Sn(2 +)-DTPA + 99mTc(7+)O4- 99m

Tc-DTPA-DG + 99mTc-DTPA + 99m
TcO2 + 99mTc(7+)O4-….
3

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 FIGURE 7. Time Reaction optimization on the 99mTc-DTPA-DG labelling (room temperature)

FIGURE 8. Time Reaction optimization on the 99mTc-DTPA-DG labelling (4 oC)

Based on this reaction, it can be seen that the reaction begins with the formation of Sn(2 +)-DTPA (Reaction 1).
Then proceed with the formation of Sn(2 +)-DTPA-DG (Reaction 2) and then the radioisotope technetium-99m is added
to form 99mTc-DTPA-DG and 3 other radiochemical impurities (Reaction 3). The 99mTc-reduced (99mTcO2) and 99mTc-
pertechnetate (99mTcO4-) radiochemical impurities can be separated using paper chromatography, but the 99mTc-DTPA
impurities cannot be separated because the Rf is the same as the 99mTc-DTPA-DG. To separate radiochemical
impurities in the form of 99mTc-DTPA, electrophoresis method was carried out. Electrophoresis is separation based
on differences in the electric charge of a compound. Therefore, in each of the optimization above, electrophoresis is
also carried out to ensure that the labelled compound formed is 99mTc-DTPA-DG instead of 99mTc-DTPA.
Electrophoresis test results showed a difference in Rf between 99mTc-DTPA and 99mTc-DTPA-DG. From the results
of the electrophoregram obtained, there was no visible impurity in the form of 99mTc-DTPA (Figure 9). This shows
that the labelled compound obtained is 99mTc-DTPA-DG instead of 99mTc-DTPA.

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 99mTcO4 99mTc-DTPA 99mTc-DTPA-DG
80
70
60
50
40

Cps
30
20
10
0
-20 -10 0 10 20
Migration (cm)

FIGURE 9. Electrophoresis result of 99mTc-DTPA-DG and blank ( 99mTc-DTPA and 99mTcO4-)

CONCLUSION
It has been obtained the optimum labelling condition of 2-deoxy-D-glucose with technetium-99m radioisotope
using the indirect method with DTPA as the bifunctional agent. The optimum 99mTc-DTPA-DG labelling conditions
were obtained at 2 mg of 2-deoxy-D-glucose, 50 µg of SnCl2 2H2O as a reducing agent, 750 µg of DTPA, 6 reaction
pH and 30 minutes of incubation time at room temperature. The radiochemical purity obtained from the optimization
results was 93.62 ± 1.04%. It is hoped that this marked compound can be developed as a tumor imaging
radiopharmaceutical.

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