Synthesis and biological evaluation of receptor-based tumor imaging agent: 99mTc-folate-glucaric acid

Article abstract of DOI:10.1016/j.ijpharm.2011.07.010

The aim of the present study was to prepare ^99mTc-folate- glucaric acid and investigate the radiopharmaceutical potential for tumors imaging that over express folate receptor. Folate-glucaric acid was synthesized and the synthesized folate conjugate was confirmed with ¹H NMR and LC-MS/MS methods. Folate-glucaric acid was labeled with ^99mTc, and its radiolabelling efficiency was found as 96 ± 2.0%. Biodistribution study of ^99mTc-folate-glucaric acid was carried out in vivo using two groups of female Albino Wistar rats: folate receptor (FR) saturated and unsaturated. Biodistribution study showed that ^99mTc-folate-glucaric acid indicated high uptake in folate receptor rich tissues such as breast, ovary and uterus. Therefore, ^99mTc-folate-glucaric acid shows good radiolabeling and biodistribution in FR organs, the radiolabeled conjugate is a reason potentially useful radiopharmaceutical for detection of FR-positive tumors.

Full text of DOI:10.1016/j.ijpharm.2011.07.010

International Journal of Pharmaceutics 416 (2011) 288–292
Contents lists available at ScienceDirect
International Journal of Pharmaceutics
journal homepage: www.elsevier.com/locate/ijpharm
Synthesis and biological evaluation of receptor-based tumor imaging agent:
99mTc-folate-glucaric acid
Mehmet Onursal, Fatma Yurt Lambrecht∗, Aykut Ozgur
Department of Nuclear Applications, Institute of Nuclear Science, Ege University, Bornova 35100 Izmir, Turkey
a r t i c l e i n f o
a b s t r a c t
The aim of the present study was to prepare 99mTc-folate-glucaric acid and investigate the radiophar-
maceutical potential for tumors imaging that over express folate receptor. Folate-glucaric acid was
synthesized and the synthesized folate conjugate was confirmed with 1H NMR and LC–MS/MS meth-
ods. Folate-glucaric acid was labeled with 99mTc, and its radiolabelling efficiency was found as 96 2.0%.
Biodistribution study of 99mTc-folate-glucaric acid was carried out in vivo using two groups of female
Albino Wistar rats: folate receptor (FR) saturated and unsaturated. Biodistribution study showed that
99mTc-folate-glucaric acid indicated high uptake in folate receptor rich tissues such as breast, ovary and
uterus. Therefore, 99mTc-folate-glucaric acid shows good radiolabeling and biodistribution in FR organs,
the radiolabeled conjugate is a reason potentially useful radiopharmaceutical for detection of FR-positive
tumors.
Article history:
Received 18 April 2011
Received in revised form 6 July 2011
Accepted 6 July 2011
Available online 18 July 2011
Keywords:
99mTc-folate-glucaric acid
Folic acid
Glucaric acid
Tumor imaging agent
Technetium-99m
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
are, increase of target/non-target tissue ratio and drug toxicity,
can be prevented at very low concentrations (Okarvi and Jammaz,
Targeted drug delivery systems are well known to be crit-
ical for therapy and diagnosis of cancer. These systems have
been successfully applied for the delivery of chemotherapeutic
and immunotherapeutic agents, genes, liposomes, proteins and
radioimaging agents into tumors (Wang et al., 1997; Sudimack
and Lee, 2000; Guo et al., 1999). Generally targeting agents with
low molecular weight are combined with chelating agents, and
labeled with suitable radionuclide (99mTc, 111In, 66/67/68Ga, 64Cu)
for nuclear medicine applications (Okarvi and Jammaz, 2006; Guo
et al., 1999; Ke et al., 2004; Jurisson and Lydon, 1999; Mindt et al.,
2008). One of the most widely used targeting agent is folic acid
(FA), which is a low molecular weight (441.4 Da) vitamin needed
for DNA synthesis and one-carbon metabolism by eukaryotic cells.
Radiolabeled folate-chelate conjugates with low molecular
weight are widely used in receptor-targeted radiopharmaceuti-
2006; Sudimack and Lee, 2000). Recently many folate-based radio-
pharmaceuticals have been synthesized and evaluated for tumor
imaging, such as 99mTc-citro-folate, 111In-DTPA-folate, 99mTc-
DTPA-CKK2-PEG-folate and 67Ga-deferoxamine-folate (Altiparmak
et al., 2010; Wang et al., 1997; Gu et al., 2010; Ke et al., 2004).
In this study a new folate conjugate, folate-glucaric acid, was
synthesized and labeled with 99mTc. The potential of radiola-
beled conjugate with low molecular weight was evaluated in
normal female rats for FR positive tumor imaging agent in nuclear
medicine.
2. Materials and methods
2.1. Materials
cal due to FA has a high-affinity for folate receptors (FR) (Kd
∼
Folic acid, dicyclohexylcarbodiimide, hydrazine hydrate and
glucaric acid were purchased from a supplier (Sigma–Aldrich
10−10 mol/L). FA binds to the folate receptors at cell surfaces via
its ␥-carboxyl group and folate conjugates internalize into the cell
by receptor mediated endocytosis (Zhang et al., 2010; Guo et al.,
1999; Okarvi and Jammaz, 2006; Sudimack and Lee, 2000; Kim et al.,
2007; Ke et al., 2003). The folate-based radiopharmaceuticals have
important advantages in imaging of wide variety of tumor types,
such as ovary, endometrial, kidney, colorectal and breast. These
Chemical
Co.,
Steinheim
am
Albuch,
Germany).
N-
hydroxysuccinimide was purchased from another supplier
(Merck Co., Darmstadt, Germany).
2.2. Synthesis of folate-hydrazide
In the first step, N-hydroxysuccinimide (NHS), which is an ester
of folic acid, was synthesized (Guo et al., 1999; Okarvi and Jammaz,
2006). For this synthesis, 1 g (2.3 mmol) folic acid was dissolved
in 50 mL dimethylsulfoxide (DMSO). Then, NHS and dicyclohexyl
∗
Corresponding author. Tel.: +90 232 3115017; fax: +90 232 388 6466.
E-mail address: fatma.yurt.lambrecht@ege.edu.tr (F.Y. Lambrecht).
0378-5173/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijpharm.2011.07.010

M. Onursal et al. / International Journal of Pharmaceutics 416 (2011) 288–292
289
carbodiimide (DCC) (1:1 ratio molar excess) were added to the
solution. The reaction was allowed to proceed for 16 h at room
temperature under stirring. The solution must be protected from
light through all reaction time. The by-product dicyclohexylurea
was removed by filtration. The NHS-folate in DMSO solution was
stored at −20 ◦C. In the second step, synthesizing folate-hydrazide
was done. NHS-folate solution was slowly added to hydrazine
hydrate (2 mL) under stirring at room temperature. The product
folate-hydrazide was converted into hydrochloride salt by adding
HCl and then precipitated with acetonitrile/diethylether (1:1 v/v).
The precipitate was centrifuged and then reprecipitated with
ethanol. The final precipitate was washed with solvent mixtures
of ethanol:diethyl ether (the ratio in mixture 1: 50/50, mixture 2:
80/20 and 100% ethanol) and the obtained precipitate was finally
lyophilized under vacuum. Then, obtained folate-hydrazide, yellow
powder, was stored at −20 ◦C.
glucaric acid, 100 ␮L tin chloride (1 mg SnCl2·2H2O/1 mL H2O),
37 MBq Na99mTcO4. The glass tube was incubated for 15 min at
room temperature. After the incubation, radiochemical product
was analyzed by RHPLC and TLRC. The sample (5 ␮L) was spotted at
the origin of three ITLC-SG strips (1.5 cm × 10 cm). The strips were
developed in different solvent systems [0.9% sodium chloride solu-
tion, methanol/water (20/80) and ethanol/ammonia/water (2/1/5)]
and then scanned using the TLC scanner.
2.6. Radiochromatography
This process was conducted with a Gelman electrophoresis
chamber. Application points on cellulose acetate strips for cath-
ode and anode poles were marked and moistened with 0.9% NaCl
solution. Each compound was set on the strip and placed into the
chamber. Applied voltage and standing time were set at 250 V and
120 min, respectively. The strips were then counted by using the
TLC scanner.
2.3. Synthesis of folate-glucaric acid
Folate-glucaric acid was synthesized as follows: glucaric acid
of 24.8 mg (0.1 mmol) was dissolved in DMSO and then 4.5 mg
(0.01 mmol) folate-hydrazide was added to glucaric acid solution.
Finally, 0.01 g (0.089 mmol) CaCl2 was added to the final solution
and stirred for approximately 1 h until the reaction was complete
(Altiparmak et al., 2010). At the end of the reaction, solution was
obtained by centrifugation. The precipitate was washed in a small
volume of water and then reprecipiated with ethanol. The pellet
was lyophilized and stored at −20 ◦C.
2.7. Stability in human serum
The serum stability of 99mTc-folate-glucaric acid was deter-
mined by incubating 200 ␮L of the labeled compound with 500 ␮L
of human serum at 37 ◦C. The sample was analyzed in the time
intervals of 30, 60, 120, 180 and 1440 min by TLRC and radioelec-
trophoresis.
2.8. Lipophilicity
The molecular structures of NHS-folate, folate-hydrazide and
folate-glucaric acid were confirmed by proton nuclear magnetic
resonance spectroscopy (1H NMR: Bruker 400 MHz spectrometer,
Berlin, Germany). LC–MS/MS analyses of NHS-folate, folate-
hydrazide and folate-glucaric acid were performed on an Agilent
6460 series system (Agilent ZORBAX Eclipse Plus C-18 narrow bore
column, 3:7 Acetonitrile:20 mM Potassium Phosphate, pH: 6.2, iso-
cratic, ambient, 0.3 ml/min).
The lipophilicity (log P) of the radiolabeled compound was
determined by using 300 ␮L radiolabeled conjugate, 99mTc-folate-
glucaric acid, added to a pre-mixed suspension of 2 mL n-octanol
in 2 mL water. The tube was vortexed for 1 h at room tempera-
ture, and then centrifuged at 3000 rpm for 5 min. Aliquots (0.5 mL)
from each phase were taken for counting. The radioactivity was
counted using a Cd(Te) detector. The partition coefficient was fixed
as partition coefficient = log10 (counts in n-octanol layer/counts in
aqueous layer). The experiments were repeated in three times and
the results were averaged.
2.4. Radiosynthesis of 99mTc-glucarate
In order to prepare radiolabeled glucarate; one mg (4 ␮mol)
glucaric acid was firstly dissolved in 500 ␮L water in a glass vial.
Secondly, 100 ␮L SnCl2·2H2O aq. solution (1 mg SnCl2·2H2O/1 mL
H2O) was added to the solution and then mixed. Finally, 99mTc-
sodium pertechnetate (37 MBq) was added and incubated at room
temperature for 15 min. The labeling efficiency of 99mTc-glucarate
was determined by thin layer radio chromatography (TLRC), using
ready plates of ITLC-SG. After developing in three different solvent
systems [0.9% sodium chloride solution, methanol/water (20/80)
solution, and ethanol/ammonia/water [(2/1/5)], the strips were
scanned on the TLC scanner (Bioscan AR-2000, Washington DC,
USA). For the RHPLC analysis, the procedures described by Alti-
parmak et al. were followed (Altiparmak et al., 2010). We used a
low pressure gradient HPLC system with LC-10ATvp quaternary
pump, UV detector (Shimadzu SPD-10ATvp, Macherey-Nagel, EC
250/4.6 Nucleodur 100-5 C18 column) and 20 ␮L loop and equipped
with a Cd(Te) detector equipped with a RAD-501 single channel
analyzer. The HPLC process was run using 0.1% TFA/acetonitrile,
0.1% TFA/water at a flow rate of 1 mL/min. The flow rate was set at
1 mL/min and the UV detector at 240 nm.
2.9. Biodistribution studies in organ tissues with saturated and
unsaturated FRs
All animal experiments were approved by the Animal Ethics
Committee of Ege University, and performed in accordance with
the published guidelines. The biodistributions of the radiolabeled
conjugate in organs with saturated and unsaturated FRs were inves-
tigated using 18 female Albino Wistar rats with weights ranging
between 130 g and 180 g. The saturated study was designed to
establish reference measurements for comparison of the radioac-
tivity obtained from the unsaturated study. Three animals were
used for each time interval and the biodistribution data from the
different organs of each rat were recorded and averaged.
FR saturation study; each animal was administered folic acid
[0.10 mg (0.23 ␮mol)] via intravenously (iv). After 30 min from folic
acid injected, 99mTc-folate-glucaric acid (10 ␮g, 2.5 GBq/␮mol) was
injected through the same route. The uptake efficiency of the radi-
olabeled conjugate was evaluated at 60, 120 and 180 min after
injection. The percent of injected dose per gram of tissue weight
(% ID/g) was then calculated considering all the measurements.
In the receptor unsaturated study, 99mTc-folate-glucaric acid
conjugate (0.015 ␮mol, 2.5 GBq/␮mol) was injected only via iv
route to the rats. The animal was sacrificed at ether atmosphere and
the organ tissues were removed and counted to establish whether
the uptake of the conjugate was specific in the receptor express-
2.5. Radiosynthesis of 99mTc-folate-glucaric acid
Synthesized folate-glucaric acid [0.5 mg (0.77 ␮mol)] was dis-
solved in 200 ␮L DMSO. 100 ␮L of the solution by taking from
the solution was diluted with 200 ␮L water. Then the following
solutions were added one by one into a glass tube: 300 ␮L folat-

290
M. Onursal et al. / International Journal of Pharmaceutics 416 (2011) 288–292
O
12
4
N
3
5
H2N
N
2
11
6
7
13
16
20
17
18
19
10
N
O
O
22
30
H
29
1
21
N
NH
OH
8
15
9
14
35
24
NH
23
25
26
27
O
28
HN
31
NH
32
O
33
34
36
OH
37
38
40
HO
39
OH
41
HO
42
43
O
44
45
HO
46
Fig. 1. Structure of folate-glucaric acid.
ing target tissue. The tissues were weighed and counted for their
radioactivities.
were measured by a Brunker 400 MHz spectrometer. The reported
chemical shifts are against TMS (tetramethylsilane). The 1H NMR
spectroscopy proved to be the most valuable tool for determin-
ing the linkage property of azide groups. Since, the azide group
can coordinate to the main structure in two possibilities, ꢀ or ˛.
The NMR spectrum of the compound (Folat-azid) in aromatic and
aliphatic regions shows 9 resonance peaks. Those 9 well-resolved
resonance peaks correspond to five different aromatic ring pro-
tons; and remaining aliphatic peaks can be attributed to–CH and
–NH groups in the chain. The position of azidation (␥ or ␣) can
be identified by the assistance of CH proton at the position of 24
(Fig. 1).
2.10. Statistical analysis
The data obtained from the biodistribution studies were eval-
uated by a statistics program (Univariate Variance Analyses and
Pearson Correlation). Probability values < 0.05 (SPSS Version 10)
were considered to be significant. A Pearson Correlation between
the organs of saturated and unsaturated animals tested for 99mTc-
folate-glucaric acid was obtained.
In the case of azidation at ␣ position, the corresponding peak
for 24, should shift to up field (around 5.50 ppm), comparing to
NMR of folic acid. In fact, the NMR spectrum of folat-azid, there was
no remarkable change for the peak at 4.47 ppm comparing to folic
acid, which means the azidation at ␥ position. NHS-folate, folate-
hydrazide and folate-glucaric acid were identified by LC–MS/MS
and their molecular masses were determined as 577, 455 and 643,
respectively.
3. Result and discussion
3.1. Characterization of folate-glucaric acid
The elementary and intermediate compounds of synthesis of
folate-glucaric acid were confirmed at each step by TLC using 2-
propanol:chloroform (7/3) mobile phase solution and then UV lamb
was used determining of Rf values. According to the Rf values,
folate-glucaric acid remained at the bottom of TLC strip (Rf = 0.0)
but other compounds were distinctly separated as listed in Table 1.
NMR and LC–MS/MS spectroscopy were used to character-
ize and confirm the synthesized folate-glucaric acid. 1H NMR
(DMSO): ı 1.84–1.96 (m, 1H, CHCH2CH2, 25), 2.03–2.10 (m, 1H,
CH2CH2C, 26), 2.65–2.67 (m, 1H, H, CHCHCH, 40), 3.84–3.87 (m,
1H, CHCHC, 42), 4.28–4.33 (q, 1H, CHCHCH, 38), 4.11–4.21 (m, 1H,
NHCHCH2, 24), 4.47 (d, J = 2.4 Hz, 1H, CCHCH, 36), 6.88 (t, J = 6 Hz, H,
CCH2NH, 13), 6.63 (d, J = 4 Hz, 2H, CCHCH, 16–20), 7.65 (d, J = 3.6 Hz,
2H, CHCHC, 17–19), 8.64 (s, CCHN, 1H, 8). Proton NMR spectra
Table 1
Rf values of folic acid, NHS-folate, folate-hydrazide, glucaric acid and folate-glucaric
acid in 2-propanol:chloroform (7/3) solution.
Compounds
Rf value
Folic acid
NHS-folate
Folate-hydrazide
Glucaric acid
Folate-glucaric acid
0.2
0.6
0.2
1.0
0.0

M. Onursal et al. / International Journal of Pharmaceutics 416 (2011) 288–292
291
1,2
1
0,8
0,6
0,4
0,2
0
60
120
180
Organs
Fig. 3. Biodistribution of 99mTc-folate-glucaric acid in unsaturated Albino Wistar
rats as a function of time.
0,4
0,35
0,3
Fig. 2. RHPLC of folat-glucaric acid, radiolabeled compund, reduced 99mTc and
99mTcO4
.
−
0,25
0,2
3.2. Quality control of 99mTc-folate-glucaric acid
60
0,15
120
0,1
The radiolabeling efficiency of 99mTc-folate-glucaric acid was
180
0,05
0
confirmed using TLRC and RHPLC methods. Rf values of 99mTc-
glucaric acid, 99mTc-folate-glucaric acid, 99mTcO4 and reduced
−
99mTc were determined as 0.77, 0.15, 1.00 and 0.09 by TLRC, respec-
tively, when mobile phase 1 (ethanol/NH3/water 2/1/5 v/v) was
used. For mobile phase 2 (methanol/water 20/80 v/v), Rf values
Organs
of 99mTc-glucaric acid, 99mTc-folate-glucaric acid, 99mTcO4 and
−
Fig. 4. Biodistribution of 99mTc-folate-glucaric acid in saturated Albino Wistar rats
as a function of time.
reduced 99mTc were 1.00, 0.20, 1.00 and 0.12, respectively. On the
other hand, when 0.9% NaCI was used as mobile phase 3, the Rf val-
ues were measured to be 0.62, 0.10, 1.00 and 0.15, respectively.
From the results of RHPLC analysis, Rt values of 99mTc-folate-
(0.02%ID/g, p < 0.05) and uterus (0.08, %ID/g, p < 0.05) at 60 min after
injection.
glucaric acid, 99mTcO4 and reduced 99mTc were found as 2.8,
−
The uptake of 99mTc-folate-glucaric acid for ovary, breast and
uterus from the saturated and unsaturated receptor biodisturibi-
tion data are shown all together for comparison from Fig. 5. As seen
in Fig. 5, the uptake decreased in ovary, when the receptor was satu-
rated. The decrease was 60% at 60 and 18% at 180 min. Data in Fig. 5
shows that the %ID/g values for uterus also decreased, when the
receptor was saturated such as 76% at 60 and 30% at 120 min. The
uptake in breast decreased, when the receptor was saturated. The
decrease for this organ was 76% at 60 and 44% at 120 min. When the
receptor saturated, the uptake in the organs decreased as reported
by Altiparmak. The percent uptake of radiolabeled compound in FR
positive organs showed a significant decrease when compared with
Altiparmak’s data (Altiparmak et al., 2010). Therefore, folate recep-
tor was blocked by unlabeled folic acid and the uptakes in ovary,
uterus and breast were determined to decrease as suggested by
Altiparmak et al. (2010). As known FRs exhibit limited expression
4.7 and 3.9 min, respectively (Fig. 2). These results indicate that
the folate-glucaric acid was successfully labeled with a high yield
(96.0 2.0%).
The serum stability result showed that 99mTc-folate-glucaric
acid remained stable during incubation at 37 ◦C up to 3 h in human
serum. Lipophilicity was determined by counting radiolabeled
folate congujate between n-octanol and water. Partition coefficient
(Log P) was calculated as −1.13 0.02 for 99mTc-folate-glucaric
acid. The result present that this compound has a water solubility
property, which is very important for in vivo distribution studies.
According to many studies, hydrophilic conjugates have showed
high affinity for folate receptors.
3.3. Biodistribution studies in Albino Wistar rats
The biodistribution evaluation of 99mTc-folate-glucaric acid
in receptor unsaturated and saturated rat groups is plotted in
Figs. 3 and 4. As seen in Fig. 3, the uptake of labeled folate-glucaric
acid in lung (0.85%ID/g), liver (6.0%ID/g, p < 0.05), small intestine
(0.97%ID/g) and spleen (0.43%ID/g) reached the highest values at
60 min after post injection (pi). Then the uptake decreased gradu-
ally in the organs with time. The corresponding values (%ID/g) in FR
including organs such as ovary, breast and uterus were determined
as 0.09 (p < 0.05), 0.07 (p < 0.05) and 0.31(p < 0.05), respectively.
The results of receptor saturated experiments are presented
Fig. 4. The data indicated that its uptake was high in lung (%ID/g:
0.15, p < 0.05), liver (%ID/g: 0.20, p < 0.05) and kidney (%ID/g: 0.34)
at 120 min and then decreased with time. According to the data,
the uptake of 99mTc-folate-glucaric acid reached maxima in folate
receptor rich tissues such as ovary (0.04%ID/g, p < 0.05), breast
0,35
0,3
0,25
0,2
0,15
0,1
0,05
breast
uterus
ovary
0
60
60
120
120
180
180
Fig. 5. Receptor saturated and unsaturated biodistrubition studies of 99mTc-folate-
glucaric acid in FR positive organs.

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M. Onursal et al. / International Journal of Pharmaceutics 416 (2011) 288–292
good radiolabeling and biodistribution in FR organs, 99mTc-
folate-glucaric acid might be potentially useful in the field of
receptor-based targeted radiopharmaceutical.
on healthy cells, but are often present in large numbers on cancer
cells. For example, FRs are over expressed on epithelial cancers of
the ovary, mammary gland, colon, lung, prostate, nose, throat, and
brain. So the use of folate ligand as targeting ligand can be poten-
tial strategy for treatment and/or monitoring of FR positive tumors
(Hilgenbrink and Low, 2005). Consequently, 99mTc-folate-glucaric
acid might be specific for ovary, uterus and breast. In the other
studies, many folate-based radiopharmaceuticals such as 99mTc-
citro-folate, 99mTc-MAG3-FA, 99mTc-MAG2-FA, 99mTc-TEPA-folate,
99mTc-DTPA-folate and 99mTc-HYNIC-folate have showed an effi-
cient uptake in the tissues including folate receptor (Altiparmak
et al., 2010; Okarvi and Jammaz, 2006; Panwar et al., 2009; Mathias
et al., 2000; Guo et al., 1999).
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In summary, 99mTc-folate-glucaric acid showed good localiza-
tion in FR rich organs such as uterus, ovary and breast and rapid
clearance from blood via urinary system.
4. Conclusions
The folate-glucaric acid was successfully labeled with 99mTc
at high radiolabelling yield (96 2.0%) and radiolabeled folate
conjugate remained stable in human serum up to 3 h. Further-
more, the conjugate was found to be neutral and hydrophilic.
As
a result of the biodistribution study conducted in the
rats showed that the 99mTc-folate-glucaric acid has signifi-
cant uptake in folate receptor rich tissues such as breast,
ovary and uterus. In conclusion, the radiolabeled conjugate shows