In vitro and in vivo evaluation of 99mTc-DO3A-EA-Folate for receptor-mediated targeting of folate positive tumors

Article abstract of DOI:10.3109/1061186X.2011.561857

Background: The cell membrane folate receptor is a potential molecular target for tumor selective drug delivery via receptor-mediated endocytosis, including delivery of radiolabeled folate-chelate conjugates for diagnostic imaging. Method: A new radiopharmaceutical, ^99mTc-1,4,7-tris (carboxymethyl)-10-(4-aminoethyl)-1,4,7,10-tetraazacyclododecane (DO3A-EA)-Folate has been synthesized introducing DO3A-EA to the γ-carboxyl group of folic acid and was characterized by different spectroscopic techniques. Cytotoxicity was determined by macrocolony and MTT assay on three different cell lines. Cell uptake studies and receptor binding assay were performed using ^99mTc-DO3A-EA-Folate. Tumor imaging was performed in KB cell line implanted tumor bearing nude mice, and uptake of the radiotracer was estimated. Results: The synthesized conjugate binds with ^99mTc at high efficiency at ambient temperature. The resulting conjugate is stable under physiological conditions for 24h after radiocomplexation. Using an in vitro receptor binding assay, the conjugate showed Kd in μM range on human tumor cell lines (KB, U-87MG and OAW). The pharmacokinetic data revealed rapid wash out of the more than 75% activity within 5min from the circulation with hepato-biliary clearance. Data from γ scintigraphic and biodistribution studies performed in KB tumor bearing nude mice revealed major accumulation of radiotracer at tumor site. High tumor uptake was shown in the tumor bearing mice; tumor to blood ratios reached 2.27±0.32 and 6.05±1.02 at 1 and 4h after post injection, respectively. Conclusion: These results suggest that ^99mTc-DO3A-EA- Folate may be clinically useful as a noninvasive radiodiagnostic imaging agent for the detection of FR-positive human cancers.

Full text of DOI:10.3109/1061186X.2011.561857

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Journal of Drug Targeting, 2011; 19(9): 761–769
© 2011 Informa UK, Ltd.
ISSN 1061-186X print/ISSN 1029-2330 online
DOI: 10.3109/1061186X.2011.561857
RESEARCH ARTICLE
In vitro and in vivo evaluation of 99mTc-DO3A-EA-Folate for
receptor-mediated targeting of folate positive tumors
Gauri Mishra1,2, Puja Panwar Hazari1, Nitin Kumar1, and Anil K. Mishra1
1Division of Cyclotron and Radiopharmaceuticals, Institute of Nuclear Medicine and Allied Sciences, Brig SK
Mazumdar Road, Delhi-110007, India and 2Department of Zoology, Swami Shraddhanand College, University of
Delhi-11007, India
Abstract
Background: The cell membrane folate receptor is a potential molecular target for tumor selective drug delivery via
receptor-mediated endocytosis, including delivery of radiolabeled folate-chelate conjugates for diagnostic imaging.
Method:
A
new radiopharmaceutical, 99mTc-1,4,7-tris (carboxymethyl)-10-(4-aminoethyl)-1,4,7,10-tetraazacyclo-
dodecane (DO3A-EA)-Folate has been synthesized introducing DO3A-EA to the γ-carboxyl group of folic acid and was
characterized by different spectroscopic techniques. Cytotoxicity was determined by macrocolony and MTT assay on
three different cell lines. Cell uptake studies and receptor binding assay were performed using 99mTc-DO3A-EA-Folate.
Tumor imaging was performed in KB cell line implanted tumor bearing nude mice, and uptake of the radiotracer was
estimated.
Results:The synthesized conjugate binds with 99mTc at high efficiency at ambient temperature. The resulting conjugate
is stable under physiological conditions for 24h after radiocomplexation. Using an in vitro receptor binding assay, the
conjugate showed Kd in μM range on human tumor cell lines (KB, U-87MG and OAW). The pharmacokinetic data
revealed rapid wash out of the more than 75% activity within 5min from the circulation with hepato-biliary clearance.
Data from γ scintigraphic and biodistribution studies performed in KB tumor bearing nude mice revealed major
accumulation of radiotracer at tumor site. High tumor uptake was shown in the tumor bearing mice; tumor to blood
ratios reached 2.27 0.32 and 6.05 1.02 at 1 and 4h after post injection, respectively.
Conclusion: These results suggest that 99mTc-DO3A-EA-Folate may be clinically useful as a noninvasive radiodiagnostic
imaging agent for the detection of FR-positive human cancers.
Keywords: Folate receptor, 99mTc-Folate, tumor imaging, bifunctional chelating agent
Introduction
and immunotherapy of ovarian cancer in clinical studies
(Weitman et al., 1992; Leamon and Reddy, 2004). ere
has been a lot of literature for folate and their conju-
gates for the application of the above studies. e reason
behind being, that folic acid, a high-affinity ligand of the
folate receptor retains its receptor binding property when
covalently derivatized via its gamma-carboxyl. Folate
conjugates have been shown to taken into receptor-bear-
ing tumor cells via folate receptor-mediated endocytosis
(Cho et al., 1997; Mathias et al., 2000; Siegel et al., 2003).
Henceforth folate conjugate presents a useful method for
e human folate receptor-α is a glycophosphatidyli
nositol-anchored membrane glycoprotein that binds
physiological folates and mediates their intracellular
transport via receptor mediated endocytosis (Antony,
1996; Toffoli et al., 1997). Meanwhile, normal tissue dis-
tribution of folate receptor is highly restricted which has
prompted search for promising folate-conjugated radio
metal chelates as potential imaging and therapeutic
agents. A monoclonal antibody against the folate recep-
tor (MOv-18) and its derivatives has been evaluated for
the scintigraphy and tomography imaging, radiotherapy
Address for Correspondence: Anil Kumar Mishra, Division of Cyclotron and Radiopharmaceutical Sciences, Department of
Radiopharmaceutical and Radiation biology, Brig. S. K. Mazumdar Road, Delhi-110054, India. Phone: +91-11-2391–4374.
Fax: +91-11-2391–9509. E-mail: akmishra@inmas.org
(Received 11 October 2010; accepted 01 February 2011)
761

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762 G. Mishra et al.
receptor-mediated drug delivery into receptor-positive
tumor cells.
and nude mice 18–24 g were used for blood clearance,
and imaging and biodistribution studies. Rabbits were
housed under conditions of controlled temperature of
22 2°C and normal diet. Athymic mice were inoculated
subcutaneous with 0.1 ml of cell suspension (1 × 106 KB
cells) into the right thigh under sterilized conditions.
Several folate receptor systems have been targeted
with low molecular weight folate-chelate conjugates.
However, only 111In-DTPA-Folate derivatives are exploited
for clinical application (Turek et al., 1993; Panwar et al.,
2004). e field of radiodiagnostic imaging has been
transitioning toward using 99mTc-based probes as it
decays more rapidly and decreases the patient’s expo-
sure time to the radioactive agent. Moreover, 99mTc-based
radiodiagnostic agents are less expensive, safer and
produce high quality images (Reddy and Low, 1998).
Consequently, several 99mTc-based folate conjugates
have been developed such as 99mTc-ethylenedicysteine,
99mTc-6-hydrazinonicotinamido-hydrazide (HYNIC) and
99mTc-DTPA-Folate (Wang and Low, 1998; Guo et al., 1999;
Ni et al., 2002; Hilgenbrink and Low, 2005).
e research has been extended using a bifunc-
tional chelate conjugated to folate; as receptor specific
radiopharmaceutical for imaging neoplastic tissues
expressing folate-binding protein, we report the syn-
thesis, characterization and radiolabeling of 99mTc with
1,4,7-tris (carboxymethyl)-10-(4-aminoethyl)-1,4,7,10-
tetraazacyclododecane folate conjugate (99mTc-DO3A-
EA-Folate).
Cell culture
Monolayer cultures of human oral carcinoma KB cells
ovarian carcinoma cells, OAW (obtained from Zoology
Department, Delhi University, India) and human malig-
nant glioma cells, U-87MG (obtained from NIMHANS,
Bangalore, India), were maintained at 37°C in a humidi-
fied CO2 incubator (5% CO2, 95% air) in DMEM (Sigma,
St. Louis, MO, Unites States) supplemented with 10%
fetal calf serum (Gibco), 50 U/ml penicillin, 50 μg/ml
streptomycin sulfate and 2 μg/ml nystatin. When the
cells were grown up to 80–90% of cellular confluence, the
fault culture cells were differentiated with trypsin-EDTA
(Gibco, Grand Island, NY).
Chemical strategy
e synthesis of Folate-DO3A-EA is a multistep process,
which is described under the following schematic steps
(Figure 1).
Scheme 1: N-Boc-2-aminoethyl bromide
Di-t-butyl-dicarbonate (9.6 g, 1.0 equivalent) in
dicholoromethane (100 ml) was added drop-
Material and methods
Chemicals
wise over 20–30 min to
a stirring of solution of
Acetonitrile (high-performance liquid chromatography
(HPLC) grade) was obtained from E. Merck Germany Ltd.,
Folic acid, N-hydroxysuccinimide, dicyclohexylcarbodi-
imide, 1,4,7-tris(carboxymethyl)-10-(4-aminoethyl)-1,
4,7,10-tetraazacyclododecane (DO3A-EA), stannous
chloride dihydrated (SnCl2.2H2O), trifluoroacetic acid
were purchased from Sigma-Aldrich Co. (St. Louis, MO,
Unites States) 99mTc was procured from Regional Center
for Radiopharmaceuticals (Northern Region), Board of
Radiation and Isotope Technology (BRIT), Department
of Atomic Energy, India.
2-bromoethylaminehydrobromide (9.02 g, 1.0 equiva-
lent) in water (50 ml) and sodium hydroxide (3.52 g, 2.0
equivalents, and 50 ml water). After 2 h, the layers were
separated and the organic layer was washed with 1N HCl
(25 ml) twice and a saturated sodium chloride (50ml)
once. e organic layer was then dried over sodium
sulfate and concentrated in vacuum to colorless oil.
Compound was purified by classical column chroma-
tography using stationary phase silica gel 60 and mobile
phase are 5% EtOAc in n-hexane. Purified compound
N-Boc-2-aminoethyl bromide 1 was concentrated under
reduced pressure at 50°C. Total yield was 8.26 g, 83.77% of
N-Boc-2-aminoethyl bromide.
Instrumentation
1
e H NMR spectra were determined by using Bruker
Avance II 400. Mass spectrum (ES+MS) was recorded on
a mass spectrometer HPLC analyses were performed
on a Waters Chromatograph efficient with 600s coupled
to a Waters 2487 photodiode array UV detector. e
C-18 RP Beckman column (5 μm, 1 mm × 1.25 cm) and
Shimadzu ODS-AQ (5 μm, 4.6 mm × 250 mm) column
were used applying elution system described in the text.
Radioimaging and biodistribution studies were done
using a planner gamma camera Hawk-Eye. e receptor
binding data were analyzed using iterative curve-fitting
program Equilibrate software from Graphpad.
ESI-MS (+): calculated for C7H14NO2Br: m/z 223.4,
225.4; found 245.9, 247.9 (M+Na). ESI-MS-MS of 247.9
13C NMR (CDCl3, 62.9 MHz) δ (ppm): 155.97 (C-2’), 80.0
(C-3’), 42.7 (C-1), 32.9(C-2) and, 28.7 (C-4’). H NMR
(CDCl3), 250 MHz, δ (ppm): 5.1 (1H of NH), 3.45-3.39 (4H
1
of C-1, C-2) 1.39 (9H of C-4’)).
Scheme 2: Synthesis of DO3A-tri-tert-butyl-ester 2 (1,4,7-
tris(carbobutoxymethyl)-1,4,7,10-tetraazacyclododecane)
2.20 equivalents of tert-butyl bromoacetate (4.0 g, 20.0
mmol) were added drop wise (in CHCl3 ~250 ml) to a
solution (in CHCl3) of one equivalent of tetraazacy-
clododecane (1.58 g, 9.1 mmol) and the solution was
stirred at room temperature for 72 h. is produced a
mixture of the two products. e free protonated cyclen
Animal models
Animal protocols have been approved by Institutional
Animal ethics Committee. New Zealand rabbits 2–3 kg
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99mTc-DO3A-EA-Folate as radiodiagnostic agents for FR-positive tumors 763
Figure 1. Chemical scheme for synthesis of Folate-DO3A-EA.
was then removed by filtration, and the resulting filtrate
was washed with water to remove remaining protonated
cyclen (which was in chloroform) and dried over anhy-
drous Na2SO4, filtered, and evaporated under reduced
pressure yielding 80% of tri-N-substituted tetraazacy-
clododecane 2, which was used for the next step without
further purification.
solid obtained after complete evaporation. Total yield
was 1.79 g, 86.05% of 10-[N-Boc-2-aminoethyl]-1, 4,7-tri
(carbobutoxymethyl)-1, 4,7,10-tetraazacyclododecane.
ESI-MS (+): calculated C33H63N5O8: m/z 657.5; found
658.5 [M+H]+, 680.4 [M+Na]. ESI-MS-MS of 658.5. 13C
NMR (CDCl3, 62.9 MHz, δ(ppm) 165.13(C-1B,C-4B,
C-7B), 159.85 (C-2’), 79.47 (C-3’), 77.12 (C-1C, C-4C,
C-7C), 49.74 (C-1A, C-7A), 48.57 (C-8, C-9, C-11, C-12),
46.89(C-2, C-6), 45.98 (C-3, C-5), 44.05 (C-10A), 43.34
ESI-MS (+): found: 515 [M+H]+; calculated for
C26H50N4O6: m/z 514. Rf = 0.64 (CHCl3/MeOH:: 5:1).
ESI-MS-MS of 515: (EI/intensity in %) (Supporting
1
(C-10B), 23.34 (C-4’) and 23.04 (C-1D, C-4D, C-7D). H
1
data) m/z = 459 (100%); 409 (88%), 347 (20%). H NMR
NMR (CDCl3), 250MHz, δ (ppm): 5.5 (1H of−NH-), 3.4-2.2
(16H of C-2, C-3, C-5, C-6, C-8, C-9, C-11, C-12, and 8H of
C-1A, C-4A, C-7A, C-10A, 2H C-10B), 1.6-1.4 (36H of C-1D,
C-4A, C-7A, C-4’).
(250 MHz, CDCl3): 1.45 (s, 27H): 2.17 to 2.81 (m, 16H):
3.33 (s, 6H): 10.01 (br, 1H). 13C NMR (CDCl3): 28.61
(C-16); 48.08 (C-9, C-11); 50.69 (C-2, C-6); 52.32 (C-8,
C-12); 53.02 (C-3, C-5); 57.21 (C-13); 80.74 (C15); 171.25,
171.41 (C-14a, C-14c).
Scheme 4: Synthesis of 1, 4,7-tris (carboxymethyl)-10-(2-
aminoethyl)-1, 4,7,10-tetraazacyclododecane 4: C16H31N5O6
e carbobutoxy derivative 3 (0.15 g, 0.19 mmol) was dis-
solved in trifluoroacetic acid (3 ml) with stirring at 0°C for
2 h followed by an additional 6h at ambient temperature.
e reaction mixture was monitored by LC-MS. After
completion, the pH was adjusted to 6 using 0.1 M NH4OAc
and water was removed to dryness. e glassy solid was
redissolved in one liter of water and the product was
lyophilized. Yield was 80%. Reverse-phase C18 HPLC: sol-
vent A, 0.1M ammonium acetate, pH 6; solvent B, MeOH;
15–65% B, 0–25 min, 65–100% B, 25–35 min; 100–15% B,
35–40 min; product peak 18 min. ESI-MS (+): found: 390
[M+H]+; calculated for C16H31N5O6: m/z 389. 1H NMR
Scheme 3: 10-[N-Boc-2-aminoethyl]-1,4,7-tri
(carbobutoxymethyl) -1, 4,7,10-tetraazacyclododecane (3)
A solution of 2 (1 g, 1 equivalent), sodium carbon-
ate (2.6 g, 10 equivalents) and ter-butyl bromoacetate
(1.64 ml, 3.5 equivalents) in acetonitrile (100 ml) was
stirred at 60–70°C for 7–8 h. Reaction was monitored
by TLC, after completion of reaction; the reaction mix-
ture was filtered and was washed with DCM (20 ml, two
times). e filtrate was then concentrated in vacuum to
obtain pale yellow oil. Compound was purified by clas-
sical column chromatography using stationary phase
silica gel 60 and mobile phase DCM: MeOH. Off white
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764 G. Mishra et al.
(D2O): 1.35 (t, 3H); 1.87 (mixed d and t, 2H); 2.41 (t, 2H);
2.51–3.29 (m, 22H), 4.07 (q, 2H). 13C NMR (D2O): 23.12
(C-18); 48.01 (C-17); 50.10 (C9, C-11); 52.4 to 52.7 (C-2 to
C-6); 56.62 (C-8, C-12); 57.21 (C-13); 173.59 (C-14).
fresh human serum at physiological conditions, i.e. at
30°C at a concentration of 100 nM/ml and then analyzed
by instant thin layer chromatography (ITLC-SG) at differ-
ent time intervals to detect any dissociation of complex.
Percentage of free pertchnetate at a particular time point
that was estimated using saline and acetone as mobile
phase, represented percentage dissociation of the com-
plex at that particular time point in serum.
Conjugation of DO3A-EA to folate
A solution of 5 (0.38 g, 0.88 mmol), folic acid (0.2 g, 0.88
mmol), NMM (0.18 ml, 1.6 mmol) and HOBt (0.13 g, 0.97
mmol) in DMF (5 ml) was stirred at 0–5°C for 15min and
then EDC (0.19 g, 0.97 mmol) was added. e reaction
mixture was stirred overnight at room temperature. e
progress of reaction was monitored by thin layer chro-
matography. After completion, the reaction mixture
was poured in water, extracted with DCM (3× 100 ml),
organic layer was dried over Na2SO4, filtered, filtrate
was evaporated under reduced pressure and purified
by column chromatography (silica gel, 10% MeOH in
dichloromethane) to give 0.32 g (55%) of 6 as dark yel-
low viscous oil. ESI-HRMS (+): calcd C29H51N7O8S: m/z
658.35926 (M+H)+; found 658.35913 (M+H)+.
Receptor binding studies
Exponentially growing cells (KB, OAW and U-87MG)
0.1 × 106 cells/PD were plated at a uniform cell density
and incubated overnight. Monolayer culture of the cell
lines were washed twice for 2 min with ice cold binding
buffer (25 mM HEPES, 10 mM MgCl2 and 1% BSA). e
cell line culture were then incubated for 40 min with
labeled DO3A-EA-Folate (varying concentration) in
the absence and presence of the 100-folds excess unla-
beled DO3A-EA-Folate for estimation of total binding
and nonspecific binding, respectively. Specific binding
was obtained by subtracting nonspecific binding from
total binding. At the end of each experiment, the cells
were washed with ice cold binding buffer three times for
3 min. e cells were lysed with 200 μl of Lysis buffer. e
cell-associated radioactivity was determined by gamma
scintillation counting. Scatchard plot analysis was done
using Equilibrate software from graph pad.
Radiolabeling and radiochemical purity
Radiolabeling was carried out as per the reference 24 and
25. 2mg solution of the compounds dissolved in water was
taken in a shielded vial and 100 µl of 1×10−2 M SnCl2.2H2O
(dissolved in N2 purged 1ml (10% acetic acid) was added
followed by addition of (<1h) freshly eluted saline solu-
−
tion of sodium pertchnetate (NaTcO4 ) (82 MBq, 200ml).
e pH of the reaction mixture was adjusted to 6.5 with
0.1M NaHCO3 solution and shook to mix the contents. e
vial was allowed to stand for 15min at room temperature.
Labeling of the compound, radiochemical purity as well
as Rf of the 99mTc complexes was determined by ITLC-SG
strips using 0.9% NaCl aqueous solution (saline) as devel-
oping solvent and simultaneously in acetone and PAW
(Pyridine, acetic acid and water in 3:5:1.5 ratio) as men-
tioned in previous literature. Each TLC was cut in 0.1cm
segments and counts of each segment were taken. By
Cytotoxicity of folate and DO3A-EA-Folate
Cytotoxicity was determined using the MTT [3-(4,
5-dimethylthiazole-2-yl)-2, 5-diphenyl-2H-tetrazolium
bromide] assay. Exponentially growing cells were
plated in a 96-well microtitre plate at a uniform cell
density of 10,000 cells/well 24 h before treatment. Cells
were treated with varying concentrations of conjugate
(mM–μM range) for various time intervals viz., 24h, 48 h,
72 h and MTT assays were performed. At the end of treat-
ment, negative control and treated cells were incubated
with MTT at a final concentration of 0.05mg/ml for 2 h at
37°C and the medium was removed. e cells were lysed
and the formazan crystals were dissolved using 150 μl of
DMSO. Optical density is measured on 150 µl of extracts
at 570 nm (reference filter: 630 nm). Mitochondrial activ-
ity was expressed as percentage of viability compared
to negative control (mean SD of triplicate cultures). %
of viability = [OD (570–630 nm) test product/OD (570–
630 nm) negative control] × 100. Percentage viability at 1
μM and 10 μM concentrations was plotted against time
for folic acid and DO3A-EA-Folate.
using this method percentage of free Na99mTcO4 , reduced
−
99mTc and the complex formed between 99mTc and folate
conjugate could be calculated. 99mTc-DO3A-EA-Folate
conjugate remained at the origin and free technetium
migrated with the solvent front in acetone.
e radiolabeled drug was injected on HPLC using
solvent gradient (eluant A, 10 mM ammonium acetate
buffer at pH 8; eluant B, acetonitrile gradient, 0 min at
4% B, 10 min at 12% B and 15 min 15% B at a flow rate of
0.5 ml/min) and fractions were collected and radioactiv-
ity was counted on automated gamma counter.
In vitro serum stability assay
Macrocolony assay
e fresh human serum was prepared by allowing blood
collected from healthy volunteers to clot for 1 h at 37°C
in a humidified incubator maintained at 5% carbon diox-
ide, 95% air. en the sample was centrifuged at 400 rpm
and the serum was filtered through 0.22 micron syringe
filter into sterile plastic culture tubes. e above freshly
prepared technetium radio complex were incubated in
Monolayer cultures of OAW and U-87MG cell lines were
trypsinized and 100–1000 cells were plated depending
upon the concentrations of the drug in 60-mm petridishes
and incubated at 37°C in 5% CO2 humidified atmosphere
for 8 days. Colonies were fixed in methanol and stained
with 1% crystal violet. Colonies containing more than 50
cells were counted.
Journal of Drug Targeting