Design and synthesis of 99mTc-citro-folate for use as a tumor-targeted radiopharmaceutical

Article abstract of DOI:10.1016/j.ijpharm.2010.08.002

Folate conjugates exhibit high affinity for folate receptor (FR) positive cells and tissues, such as those in tumors, making them attractive candidates and of interest in diagnostic tumor imaging. The aim of study was to synthesize a novel radiopharmaceut

Full text of DOI:10.1016/j.ijpharm.2010.08.002

International Journal of Pharmaceutics 400 (2010) 8–14
Contents lists available at ScienceDirect
International Journal of Pharmaceutics
journal homepage: www.elsevier.com/locate/ijpharm
Design and synthesis of 99mTc-citro-folate for use as a tumor-targeted
radiopharmaceutical
Burcu Altiparmak, Fatma Yurt Lambrecht∗, Elif Bayrak, Kubra Durkan
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
Article history:
Received 1 April 2010
Received in revised form 24 July 2010
Accepted 2 August 2010
Available online 7 August 2010
Folate conjugates exhibit high affinity for folate receptor (FR) positive cells and tissues, such as those
in tumors, making them attractive candidates and of interest in diagnostic tumor imaging. The aim
of study was to synthesize a novel radiopharmaceutical based folate conjugate, 99mTc-citro-folate, and
to evaluate its efficiency as a targeted agent for imaging tumors that over express FR. TLC, HPLC 1H
NMR and LC–MS/MS methods were used to check and confirm the synthesized citro-folate. Citro-folate
was labeled with Tc-99m with high labeling efficiency (97 1.0%). Biodistribution of the radiolabeled
conjugate 99mTc-citro-folate was investigated in vivo using two groups of rats: FR saturated and unsatu-
rated. These experiments showed high uptake of 99mTc-citro-folate in FR rich tissues and demonstrated
its sensitivity and specificity in imaging ovary and uterus. Based on the demonstrated good radiola-
beling and biodistribution properties, the compound 99mTc-citro-folate may potentially be used as a
radiopharmaceutical agent for imaging the FR-positive tumors.
Keywords:
99mTc-citro-folate
Folic acid
Citric acid
Tumor imaging agent
99mTc
© 2010 Elsevier B.V. All rights reserved.
capability to be labeled with radioisotope Technetium 99m (99mTc),
we explored the benefits of using citric acid as a ligand to folate
1. Introduction
(Ercan et al., 1992; Sager et al., 1977). 99mTc is commonly cho-
sen for imaging in diagnostic nuclear medicine due to it is ideal
energy (Eꢀ = 140 keV), low radiation dose, long half-life of 6 h, and
wide commercial availability (Baum, 1975; Miguel et al., 2006;
Subramanian et al., 1975). In the following, we describe the design
and synthesis of the radiopharmaceutical 99mTc-citro-folate, and
biodistribution studies aimed at evaluating its potential use for FR-
targeted tumor imaging agent in nuclear medicine using in vitro
and in vivo experiments.
Folate receptor (FR) frequently is overexpressed in a wide
range of tumor types, thereby making it an attractive target for
tumor-selective radioimaging. It has specifically been identified
as a biomarker for detecting ovarian carcinomas and a number
of folate-based radiopharmaceuticals have been developed and
evaluated for this purpose (Kim et al., 2007; Müler et al., 2006,
2008; Sudimack and Lee, 2000; Siegel et al., 2003; Guo et al., 1999;
Wang et al., 1997). Past research efforts resulted in many radio-
labeled folate conjugates for FR-mediated diagnostic imaging of
other tumors (Kim et al., 2007; Müler et al., 2006, 2008; Sudimack
and Lee, 2000; Siegel et al., 2003; Guo et al., 1999; Wang et al.,
1997; Okarvi and Jammaz, 2006; Ke et al., 2003, 2004): 99mTc-PEG-
folate, 67Ga-DF-folate, 111In-DTPA-folate, and 99mTc-HYNIC-folate.
But, so far, the potential of using citro-folate (short for citric acid
conjugated to folate) as a chelating agent for tumor imaging is still
remain unknown. A folate-based radiopharmaceutical is synthe-
sized at low molecular weight. A low weight compound is easily
uptaken into the cell by the process of receptor-mediated endo-
cytosis. This makes the compound more favorable in terms of its
pharmacokinetic properties as compared to much larger radiola-
beled antibody (Jurisson and Lydon, 1999). In this study, because
of its low molecular weight and also its clinically demonstrated
2. Materials and methods
2.1. Materials
Folic acid, dicyclohexylcarbodiimide and hydrazine hydrate
were purchased from
a supplier (Sigma–Aldrich Chemical
Co., Steinheim am Albuch, Germany). Citric acid and N-
hydroxysuccinimide were purchased from another supplier (Merck
Co., Darmstadt, Germany).
2.2. Synthesis of folate-hydrazide
First, N-hydroxysuccinimide (NHS) ester of folic acid was syn-
thesized (Guo et al., 1999; Okarvi and Jammaz, 2006). 1 g (2.3 mmol)
of folic acid was dissolved in 50 mL dimethylsulfoxide (DMSO). A
1:1 ratio molar excess of NHS and dicyclohexyl carbodiimide (DCC)
∗
Corresponding author. Tel.: +90 23233434000/5017; fax: +90 2323886466.
E-mail address: fatma.yurt.lambrecht@ege.edu.tr (F.Y. Lambrecht).
0378-5173/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijpharm.2010.08.002

B. Altiparmak et al. / International Journal of Pharmaceutics 400 (2010) 8–14
9
Fig. 1. Synthesis of folate-hydrazide.
were then added to the solution. The reaction was allowed to pro-
ceed for 16 h at room temperature under stirring and shielding
from light. The by-product dicyclohexylurea was removed by fil-
tration. The DMSO solution of the NHS-folate was stored at −20 ◦C
until it was used. Then, to synthesize folate-hydrazide (Fig. 1), 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 pre-
cipitated with acetonitrile/diethylether 1:1. The precipitate was
centrifuged, redissolved in 1 mL water and then reprecipitated
with ethanol. The final pellet was washed with solvent mixtures
of ethanol:diethyl ether (the ratio in mixture 1: 50/50, mixture 2:
80/20 and 100% ethanol) and then lyophilized into a yellow pow-
der under vacuum. Then, the yellow powder, folate-hydrazide, was
stored at −20 ◦C.
(0.01 mmol) folate-hydrazide was added to prepare citric acid solu-
tion. The final solution was added 0.0056 g (0.05 mmol) CaCl2
and stirred for approximately 1 h until the reaction was complete
(Gourrierec et al., 2008). The resulting pellet was then washed three
times by dissolution in a small volume of water followed by pre-
cipitation with ethanol. The pellet was lyophilized and stored at
−20 ◦C (Fig. 2).
The molecular structures of NHS-folate, folate-hydrazide and
citro-folate were confirmed by proton nuclear magnetic resonance
spectroscopy (1H NMR: Bruker 400 MHz spectrometer, Berlin,
Germany) and liquid chromatography–mass–mass spectroscopy
(LC–MS/MS: Agilent LC-MSD SL ion trap (Agilent HPLC 1100 binary
pump, degasser, autosampler, column oven).
2.4. Radiochemical synthesis of 99mTc-citrate
2.3. Synthesis of citro-folate
The following procedure was used for radiolabeling. First, 0.01 g
(0.05 mmol) citric acid (Brothers Chem. Co., USA) was dissolved
in 2 mL water in a glass vial. The pH was adjusted to 5 with
0.5 N NaOH; 200 ␮L SnCl2 2H2O aq. solution (1 mg/mL) was added
Citro-folate was synthesized as follows. Citric acid, of amount
0.0192 g (0.1 mmol), was dissolved in DMSO, then 0.0045 g

10
B. Altiparmak et al. / International Journal of Pharmaceutics 400 (2010) 8–14
Fig. 2. Synthesis of citro-folate.
2.7. Stability of 99mTc-citro-folate in human serum
and mixed well. Next, 2 mL generator (Amersham International
Inc., Amersham, UK) eluate containing 370–555 MBq of 99mTc as
pertechnetate was added and left to react at room temperature
for 10 min. The labeling efficiency of 99mTc-citrate was determined
by Radio-Thin-Layer-Chromatography (RTLC), using ready plates
of ITLC-SG and Radio High Performance Liquid Chromatography
(RHPLC) using HPLC (Shimadzu, Japan). After developing in one of
the three different solvent systems (0.9% sodium chloride solution
and acetone and ACD [citrate-dextrose buffer solution (Eczacibasi-
Baxter, Turkey)], the strips were scanned on the TLC scanner
(Bioscan AR-2000, Washington DC, USA). For the RHPLC analysis,
we followed the procedures described by Guo et al. (1999). 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. The UV detector was settled at 240 and 280 nm.
In vitro stability of 99mTc-citro-folate was determined by incu-
bating 300 ␮L of the labeled compound with 600 ␮L of human
serum at 37 ◦C. The solution was then analyzed in time intervals
of 15, 60, 180, 240 and 1440 min by radioelectrophoresis and RTLC.
2.8. Lipophilicity
The lipophilicity (log P) of the radiotracer was measured as fol-
lows: 100 ␮L of the radiolabeled conjugate, 99mTc-citro-folate, was
added to a premixed suspension of 3 mL n-octanol in 3 mL water.
The resulting solution was mixed for an hour at room temperature
and allowed to stand for the formation of phases. Solution of 100 ␮L
of each phase was removed and radioactivity was counted using a
Cd(Te) detector. The experiments were conducted in triplicate and
the measurements were averaged.
2.9. Biodistribution studies in organ tissues with saturated and
unsaturated FRs
2.5. Radiochemical synthesis of 99mTc-citro-folate
All animal experiments were approved by the Animal Ethics
Committee of Ege University, and performed in accordance with
the published guidelines. The saturated study was designed to
establish reference measurements for comparison of the radioac-
tivity obtained from the unsaturated study. The biodistributions
of the radiolabeled conjugate in organs with saturated and unsat-
urated FRs were investigated using 18 female Albino Wistar rats
with weight range between 100 and 150 g. The rats were randomly
divided into 6 groups, each with 3 rats, for the FR saturated and
unsaturated (control) studies at 3 different time points (60, 120 and
180 min). The biodistribution readings from the different organs of
each rat were recorded and the results were group-averaged to
obtain final measurements.
For the FR saturation study; each animal was administered
folic acid, 300 ␮g (0.68 ␮mol) intraperitonally. After 30 min of folic
acid administration, 99mTc-citro-folate was injected through the
same route. The uptake efficacy of the radiolabeled conjugate
was assessed at 60, 120 and 180 min post-injection to determine
whether the uptake of the conjugate was specific in the recep-
tor expressing target tissue. Lower radioactivity implied saturation
of the FRs by the prior injected unlabeled folic acid and hence
0.5 mg (0.83 ␮mol) of synthesize citro-folate was dissolved
in 1 mL DMSO. The following solutions were sequentially added
to a glass tube: 300 ␮L citro-folate, 100 ␮L tin chloride (1 mg
SnCl2.2H2O/1 mL H2O), 37 MBq 99m Tc-sodium pertechnetate. The
glass tube was incubated for 20 min. The radiochemical prod-
uct was then analyzed by RTLC and RHPLC for purity. Briefly,
5 ␮L samples were spotted at the origin of two ITLC-SG strips
(1.5 cm × 10 cm). The strips were developed in two different sol-
vent systems (0.9% sodium chloride solution and acetone) and then
scanned using the TLC scanner.
2.6. Radioelectrophoresis procedure
This process involved a Gelman electrophoresis chamber. Cath-
ode and anode poles and application points on cellulose acetate
strips were marked and moistened with 0.9% NaCl solution. Each
compound was set on one strip and placed in the chamber. Standing
time and applied voltage were set at 90 min and 250 V, respectively.
The strips were then counted using the TLC Scanner.

B. Altiparmak et al. / International Journal of Pharmaceutics 400 (2010) 8–14
11
Table 1
interpreted as the indicator of the specificity of the radiolabeled
Rf values of folic acid, NHS-folate, folate-hydrazide, citric
acid and citro-folate compounds in 2-propanol:chloroform
(7/3) solution.
conjugate. The percent of radioactivity per gram of tissue weight
(%ID/g) was then calculated from the measurements.
For the receptor unsaturated study, we did not adminis-
ter folic acid, but 99mTc-citro-folate conjugate (specific activ-
ity = 1.32 MBq/mmol), 20 ␮g (0.03 ␮mol), was injected only
through the same intraperitoneal route to the rat. The animal was
sacrificed and the organ tissues were again harvested at 60, 120 and
180 min of the injection. All tissues were weighed and counted for
their radioactivities as has been done in the saturation study. The
difference between the radioactivities of the sample organ from
the saturated and unsaturated studies was used as the indicator of
the binding efficiency of the manufactured conjugate in that target
tissue.
Compounds
Rf values
Folic acid
0.2
0.6
0.2
0.7
0.8
NHS-folate
Folate-hydrazide
Citric acid
Citro-folate
CH2NH, 24), 4.49 (d, J = 4.8 Hz, 2H, NHCH2CH, 12), 6.63 (d, J = 9.2 Hz,
2H, CHCHC, 15–19), 6.91 (m, CHNHC, 1H, 23), 7.67 (d, J = 8.8 Hz, 2H,
CCHCH, 16–18), 8.03 (m, CNHCH2, 1H, 13), 8.64 (s, CCHN, 1H, 13).
The spectrum was analyzed using the spectrometer’s software.
The reported chemical shifts are against TMS (tetramethylsilane).
The proton spectroscopy has been proven to be the most valuable
tool for determining the linkage property of azide group. Since,
the azide group can coordinate with the main structure in two
possiblities, ␥ or ␣ position (Fig. 4). The spectrum of the com-
pound folate-hydrazide in aromatic and aliphatic regions shows
9 resonance peaks. These well-resolved peaks correspond to five
different aromatic ring protons, and the remaining aliphatic peaks
are attributed to –CH and –NH groups in the chain. The position
of azidation (␥ or ␣) is identified with the assistance of CH proton
at position 24 (Fig. 4). Azidation at ␣ position shifts the peak at 24
upwards by about 5 ppm, when compared to the spectrum of folic
acid. But, in our spectrum, no remarkable change was observed
around this peak. Differently from folic acid, the spectrum of
folate-hydrazide exhibited a peak at 4.33 ppm, which suggested
azidation at ␥ position. ␥ position provides a binding site for folic
acid to FR, and hence it is a desirable feature of a synthesized
conjugate designed to target FR. In this regard, our compound
folate-hydrazide shares this feature and plays a significant role
in increasing the affinity of citro-folate towards FR. Collectively,
the results from the different evaluation methods for defining
molecular structure clearly demonstrated the successful synthesis
of the citro-folate conjugate using our experimental protocols.
2.10. Statistical analysis
Differences in the mean values of measured activities were
evaluated statistically by SPSS 10 programme (Univariate Variance
Analyses and Pearson Correlation). Probability values <0.05 were
considered to be significant. A Pearson Correlation between the
organs of saturated and unsaturated animals, testing for 99mTc-
citro-folate was obtained.
3. Results and discussion
3.1. Characterization of citro-folate conjugate
The intermediate compounds (NHS-folate and folate-hydrazide)
were in part synthesized during the process of manufacturing citro-
folate were confirmed one-by-one by TLC and HPLC methods. Rf
values from the TLC analysis of each compound were distinctly sep-
arate as listed in Table 1. HPLCs in Fig. 3 also indicated different
retention times (Rt) for these compounds.
The products NHS-folate (N-hydroxysuccinimide-folate),
folate-hydrazide and citro-folate were identified by LC–MS/MS
and had molecular masses 527, 455 and 615, respectively. Char-
acterization of citro-folate conjugate by 1H NMR spectroscopy at
400 MHz yielded its structure 1H NMR (DMSO): ı 2.05–1.82 (m, 2H,
CH2CH2CH, 25), 2.41–2.15 (m, 2H, CCH2CH2, 26), 4.32–4.28 (q, 1H,
Fig. 3. HPLC chromatographies of folic acid, NHS-folate, folate-hydrazide, citric acid and citro-folate compounds.
Fig. 4. Structure of folate-hydrazide and folic acid.

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B. Altiparmak et al. / International Journal of Pharmaceutics 400 (2010) 8–14
radiolabeled conjugate demonstrated that human serum contained
approximately 95.4% of 99mTc-citro-folate as an intact complex
within 180 min after introducing it to the serum.
3.3. Biodistribution results
In vivo biodistribution studies involved experiments with rats
in receptor saturated and unsaturated groups, and the results
obtained from these studies were summarized in Figs. 6 and 7.
According to the data in Fig. 6, the uptake of 99mTc-citro-folate
in small intestine, large intestine, stomach, uterus and ovary was
maximum at 60 min after the administration of the compound. The
corresponding values were measured in the order of %ID/g: 0.10
(p < 0.05), 0.7, 0.07, 0.14 and 0.07, respectively. On the other hand,
the uptake at 180 min decreased in the organs, [large intestine
(%ID/g = 0.06 (p < 0.05)) and stomach (%ID/g = 0.02)]. The clearance
of the radiolabeled folate conjugate was slower in the target organs
equipped with FR than the non-targeted organs. While the read-
ings in ovary and uterus were %ID/g 0.07 and 0.14 (p < 0.05) at
60 min, these uptake values decreased to %ID/g 0.04 (p < 0.05) and
0.02 at 180 min, respectively. These results together demonstrated
that the uptake of 99mTc-citro-folate was FR mediated. This was
further supported by the positive results obtained from the recep-
tor saturated experiments as summarized in Fig. 7. According to
the figure, the radiolabeled citro-folate uptake reached maxima
at 60 min in the following organs: small intestine (%ID/g = 0.09),
large intestine (%ID/g = 0.06) (p < 0.05), stomach (%ID/g = 0.09), kid-
ney (%ID/g = 0.06), ovary (%ID/g = 0.06) and uterus (%ID/g = 0.07). In
the order of organs presented, the corresponding %ID/g values at
180 min were all lower in magnitude: 0.04, 0.05, 0.03, 0.10, 0.02
and 0.02, respectively.
−
Fig. 5. RHPLC chromatograms of 99mTc-citro-folate, 99mTcO4 and H-R 99mTc.
3.2. Quality control of 99mTc-citro-folate
Theradiolabelingefficiencyoftheradiolabeled conjugate99mTc-
citro-folate was also confirmed using methods RTLC and RHPLC. The
data obtained by examining the chromatograms showed that we
were able to produce radiolabeled citro-folate with an efficiency
of 97 1.0%. The Rf values from RTLC for the other compounds
99mTc-citrat, 99mTc-citro-folate, 99mTcO4−, H-R 99mTc were 0.1,
0.03, 0.99 and 0.00, respectively, when acetone was used as the
developing solution. But, when ACD was used as the solution, the
corresponding measures were 0.97, 0.03, 0.96 and 1.00, respec-
tively. In consistent with these results, Rt values from RHPLCs in
Fig. 5 were 2.8, 3.78 and 5.31 min for 99mTc-citro-folate, reduced
Tc-99m and pertechnetate, respectively. These results together
provided the evidence that we were also successful in radiolabeling
the conjugate of our interest. In vitro study on the stability of the
Fig. 6. Receptor unsaturated biodistrubition results of 99mTc-citro-folate conjugate in Albino Wistar rats.
Fig. 7. Receptor saturated biodistrubition results of 99mTc-citro-folate conjugate in different organs at 60, 120 and 180 min after its injection.

B. Altiparmak et al. / International Journal of Pharmaceutics 400 (2010) 8–14
13
Hydrophilic conjugates, such as 99mTc-citro-folate, remains
longer in blood due to its negative charge. The increased cir-
culation time leads to a rapid clearance of the conjugate from
the blood by kidneys. These findings were consistent with the
observations made with other hydrophilic compounds 99mTc-
MAG3-MTX and 99mTc-MAG3-FA in the literature (Okarvi and
Jammaz, 2006). In previous study performed with 99mTc-HYNIC-
folate, Guo et al., suggested that hydrophilic radiopharmaceuticals
exhibit special feature of fast clearance from blood pool through
kidney. This property makes these radiopharmaceuticals ideal
candidates for using in scintigraphic imaging (Guo et al., 1999).
So far, several radiolabeled folate conjugates; 99mTc-PEG-folate
(Kim et al., 2007) 67Ga-DF-folate (Sudimack and Lee, 2000), 111In-
DTPA-folate (Wang et al., 1997), 99mTc-HYNIC-folate (Guo et al.,
1999), 99mTc-EC-20 (Reddy et al., 2004), 18F-FBA-folate (Bettio
et al., 2006), etc. (has been synthesized using different chela-
tion agents and evaluated for tumor imaging. But we labeled
firstly citro-folate with 99mTc, and the efficiency of the radiola-
beled conjugate was of 97 1.0%, and evaluated in normal female
rats.
Fig. 8. Receptor saturated and unsaturated biodisturibition studies of 99mTc-citro-
folate in ovary tissue.
Wang et al. (1997) indicated that the tumor to blood ratios of
111In-DTPA-folate at 4 h post i.v. administration was 82 to 1. Siegel
et al. (2003) confirmed that 111In-DTPA-folate is safe, and possi-
bly effective, for imaging of ovarian malignancy, perhaps allowing
presurgical differentiation between malignant and benign ovarian
masses. In performed study by Guo et al., the receptor binding
property of 99mTc-HYNIC-folate was studied in cultured KB (a
human oral cancer cell line) cells. It is shown that FR-mediated
uptake was ∼300 times the non-specific binding in the presence
of 1 mM free folic acid (Guo et al., 1999). Sudimack and Lee (2000)
demonstrated that 67Ga-DF-folate was shown to have 100 times
greater uptake in FR-positive oral carcinoma KB cells than the
non-targeted 67Ga-DF. On the other hand, in cell culture study per-
formed by Okarvi and Jammaz (2006), it was proved more than
affinity to FR 99mTc-MAG2-folate to 99mTc-MAG3-folate. In another
study related to PEG-folate results show that the images were
recorded at 30 min, 1, 2, 3 and 4 h after administration of 99mTc-
PEG-folate. Until 4 h, the images especially showed specific binding
to the tumor and strong uptake in comparison with normal mus-
cle. Moreover, the tumor-to-normal muscle ratio reached 4.3 at
4 h. In the competition study, the tumor uptake was blocked by
the co-injection of free folic acid, and the image indicates their
conjugate is not taken up via the FR for tumor localization, and
the tumor-to-normal muscle ratio was under 1.2 at 4 h (Kim et al.,
2007).
Fig. 9. Receptor saturated and unsaturated biodisturibition studies of 99mTc-citro-
folate in uterus tissue.
The temporal uptake of 99mTc-citro-folate for ovary and uterus
from the saturated and unsaturated receptor studies are depicted
all together for side-by-side comparison in Figs. 8 and 9. Data in
Fig. 8 shows that the uptake in ovary decreased when the receptor
was saturated. The decrease was 11, 41 and 41% at 60, 120 and
180 min, respectively. The %ID/g values for uterus also decreased
when the receptor was saturated as seen in Fig. 9. The uptake for
this organ was 47, 31 and 26% at 60, 120 and 180 min, respectively.
The uptakes in uterus and ovarian were observed to decrease in
saturated study (Figs. 8 and 9). Non-radiolabeled folic acid is able
to reduce uptake in receptor rich tissues, such as the uterus and
ovary. These results indicated that radiolabeled conjugate might
be specific for ovary and uterus.
Our results indicated that the uptake is increased in kidney
with time as shown in Fig. 6, suggesting shorter clearance time
for the compound to be extracted from blood. To understand the
origin of this behavior we performed additional studies to examine
the lipophilicity of our conjugate. We used paper electrophoresis
method to test the polarity of electric charge and also radiolabeling
efficiency. The results from this test showed that both 99mTc-
citric acid and 99mTc-citro-folate assumed negative charges. The
lipophilicity analysis indicated low lipophility for the 99mTc-citro-
folate conjugate. But, as shown in Table 2, the measured value was
higher than the theoretical estimates calculated for folic acid or
citro-folate. This difference may be attributed to the incomplete-
ness of the assumptions that led to the theoretical derivations.
Given these vast amounts of previous studies, our compound
99mTc-citro-folate is an incremental addition to the extensive
library of folate conjugated radiopharmaceuticals and the radiola-
beled conjugate showed localization in the tissues including folate
receptor as uterus and ovary. The result demonstrated the ability
of 99mTc-citro-folate in the express FRs tissues.
4. Conclusion
Our results in this study demonstrated that it is possible
to successfully label citro-folate with 99mTc at high labeling
efficiency (97 1.0%) and the resulting radioactive conjugate
99mTc-citro-folate remains stable in serum and is feasible for
in vivo use. The compound has greater affinity to those cells
expressing FR as indicated by the accumulation of the com-
pound at the FR-mediated organs such as uterus, ovarian and
breast. The results of this study are significantly encouraging to
bring about further evaluation of the radiolabeled folate con-
jugate as a possible folate receptor-positive tumors imaging
agent.
Table 2
Log P values of the compounds folic acid, citric acid, citro-folate and 99mTc-citro-
folate.
Compound
Theoretical log P
Experimental log P
Folic acid
Citric acid
−2.32 0.66
0.63 0.40
−2.35 0.81
–
–
–
–
Citro-folate
99mTc-citro-folate
−0.96 0.39

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B. Altiparmak et al. / International Journal of Pharmaceutics 400 (2010) 8–14
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The authors gratefully acknowledge the financial support
received from Department of Scientific Projects at Ege University,
Izmir, Turkey.
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