Biological evaluation of avidin-based tumor pretargeting with DOTA-Triazole-Biotin constructed via versatile Cu(I) catalyzed click chemistry

Article abstract of DOI:10.3109/1061186X.2010.504269

Background: The biotin-avidin interaction remains a gold standard for the two-step pretargeting approach to image tumor sites. We aim to develop two-step pretargeting systems utilizing ^99mTc labeled biotin functionalized macrocyclic chelating agents synthesized using the highly efficient Cu(I) catalyzed azide-alkyne cycloaddition for potential radioimaging applications. Methods: A facile synthesis of DOTA-Triazole-Biotin, radiocomplexation with ^99mTc and the pretargeting protocol is described. The synthesis features Cu(I) catalyzed click conjugation between biotinylated azide and propynyl functionalized DO3A. ^99mTc radiolabeling was performed to detect the accumulation of avidin as the pretargeting agent. Cytotoxicity was determined using the trypan blue exclusion assay, macrocolony, and MTT assay. Cell uptake studies were performed using radiolabeled DOTA-Triazole-Biotin and compared with avidin treated cells for 2 h. Tumor imaging was performed in U-87MG cell line implanted tumor bearing nude mice and uptake of the radiotracer was estimated. Results: All compounds have been successfully characterized by NMR and MS spectroscopy. More than 96% radiolabeling efficiency was obtained and the radioconjugate exhibited sufficient stability under physiological conditions. Conclusion: To summarize, a new candidate for avidin based two-step pretargeting of tumors has been synthesized and evaluated for potential imaging and diagnostic applications. The chelate possesses high stability under physiological conditions, exhibits effective interaction with its avidin target, and low nonspecific retention in vivo.

Full text of DOI:10.3109/1061186X.2010.504269

Journal of Drug Targeting, 2011; 19(6): 418–426
© 2011 Informa UK, Ltd.
ISSN 1061-186X print/ISSN 1029-2330 online
DOI: 10.3109/1061186X.2010.504269
RESEARCH ARTICLE
Biological evaluation of avidin-based tumor pretargeting
with DOTA-Triazole-Biotin constructed via versatile Cu(I)
catalyzed click chemistry
Jasleen Kaur Uppal1,2, Raunak Varshney1, Puja Panwar Hazari1, Krishna Chuttani1, Narender Kumar
Kaushik2, and Anil Kumar Mishra1
1Division of Cyclotron and Radiopharmaceutical Sciences, Institute of Nuclear Medicine and Allied Sciences, Delhi,
India, and 2Department of Chemistry; University of Delhi, Delhi, India
Abstract
Background: The biotin-avidin interaction remains a gold standard for the two-step pretargeting approach to
image tumor sites. We aim to develop two-step pretargeting systems utilizing 99mTc labeled biotin functionalized
macrocyclic chelating agents synthesized using the highly efficient Cu(I) catalyzed azide–alkyne cycloaddition for
potential radioimaging applications.
Methods: A facile synthesis of DOTA-Triazole-Biotin, radiocomplexation with 99mTc and the pretargeting protocol
is described. The synthesis features Cu(I) catalyzed click conjugation between biotinylated azide and propynyl
functionalized DO3A. 99mTc radiolabeling was performed to detect the accumulation of avidin as the pretargeting
agent. Cytotoxicity was determined using the trypan blue exclusion assay, macrocolony, and MTT assay. Cell uptake
studies were performed using radiolabeled DOTA-Triazole-Biotin and compared with avidin treated cells for 2h.
Tumor imaging was performed in U-87MG cell line implanted tumor bearing nude mice and uptake of the radiotracer
was estimated.
Results: All compounds have been successfully characterized by NMR and MS spectroscopy. More than 96%
radiolabeling efficiency was obtained and the radioconjugate exhibited sufficient stability under physiological
conditions.
Conclusion:To summarize, a new candidate for avidin based two-step pretargeting of tumors has been synthesized and
evaluated for potential imaging and diagnostic applications. The chelate possesses high stability under physiological
conditions, exhibits effective interaction with its avidin target, and low nonspecific retention in vivo.
Keywords: Tumor/nontumor ratio, dipolar cycloaddition, tetrazacyclododecane, receptor binding, tumor imaging
Introduction
glycolipids) are expressed at various levels on the sur-
Among the radiometal-labeled compounds with tumor-
targeting properties, biotin derivatives carrying mac-
rocyclic chelators, have disclosed new horizons in the
diagnosis and radioimmunotherapy (RIT) of cancer in
conjunction with the administration of avidin or strepta-
vidin proteins (Sabatino et al., 2003). Avidin-biotin
pretargeting has achieved promising clinical results in
imaging and treating various tumors. Lectins (proteins
that bind specific sugar molecules on glycoproteins and
face of tumor cells. Conjugation of cytotoxic agents to
glycoproteins recognized by lectins could be useful in
the treatment of tumors. Avidin (a highly glycosylated,
positively charged protein found in egg white) contains
terminal N-acetylglucosamine and mannose residues
that bind to lectins (Yao et al., 1998). In this study, we
tested the ability of avidin, labeled through conjugation
to radioactive biotinylated chelate to target intraperi-
toneal tumors. e high specificity and strong affinity
Address for Correspondence: Dr. Anil Kumar Mishra DCRS, Institute of Nuclear Medicine and Allied Sciences, Brig. S. K. Mazumdar Road,
Delhi-110054, India. E-mail: akmishra@inmas.drdo.in
(Received 05 March 2010; revised 01 June 2010; accepted 11 June 2010)
418

Evaluation of DOTA-Triazole-Biotin based tumor pretargeting 419
(Kd = 10–15 M) of avidin for biotin plays a significant role
in the two-step pretargeting protocol based on admin-
istration of the avidin antibody conjugate followed by
the radioactive biotin chelate (Gonza´lez & Murphy,
2003; Sakahara & Saga, 1999). e interaction of biotin
with avidin has been shown to be stable in vivo and is
essentially irreversible (Krivickas et al., 2007). Multistep
avidin/biotin methods result in enhanced tumor-to-
nontumor ratios by placing the radiolabel on a small
molecule designed to clear from the circulation and
whole body rapidly compared with the conventional
“one step” approach with radiolabeled monoclonal
antibodies (Hainsworth et al., 2005). Another advan-
tage of biotin-avidin for pretargeting is the tetrameric
architecture (four fold valency) of this protein for biotin,
providing the potential of modest signal amplification
at the tumor site (Chen, 2008). ey based their initial
pretargeting studies on a DTPA-biocytin analogue and
subsequently described the preparation of a DOTA-
biotin analogue. Mather and coworkers developed
DOTA-Lysine-biotin conjugate as an effector molecule
for pretargeted radionuclide therapy.
To simplify the synthesis of biotinylated chelates,
the versatile class of Cu(I) catalyzed click chemistry
was employed. A novel alkyne-functionalized diethyl-
enetriaminetetraacetic acid (DTTA) chelate has been
developed and coupled to a per-azidocyclodextrin core
via click chemistry (Bryson et al., 2008). e click chem-
istry-based synthesis of bistriazole-based polyamin-
ocarboxylic acid derivatives has been elaborated in
literature (Camp et al., 2008). Click conjugation refers
to a Cu(I) catalysed Huisgen 1,3-dipolar cycloaddi-
tion of azides and alkynes, providing 1,4-disubstituted
1,2,3-triazole ring in high yield with minimal work-up
and purification of reaction products (Chittepu et al.,
2008). It offers in situ generation of Cu(I) salts needed
for the catalysis from the stable Cu(II) sulfate/sodium
ascorbate redox system as Cu(I) catalysis dramatically
improves regioselectivity to afford the 1,4-regioisomer
exclusively and increases the reaction rate up to 107
times (Bock, 2006).
moment of the triazole system is larger than that of the
amide bond, its hydrogen bonding donor and acceptor
properties are more marked than those of an amide bond
(Knox et al., 2000).
Extensive interest in the rational design and synthe-
sis of organic polydentate ligands based on 1, 4, 7, 10-
tetraazacyclododecane tetraacetic acid (DOTA) stems
mainly from their complexes with radioactive metals
(64Cu, 99mTc) to be used in potential diagnostic/therapeu-
tic applications. ese metal complexes find widespread
utility as a consequence of their robust steric and elec-
tronic properties and well-defined coordination chemis-
try. e set of coordinating donor atoms (four nitrogens
and four oxygens) wraps around a lanthanide (III) ion in
a very efficient way to yield complexes endowed with a
very high thermodynamic and kinetic stability. On aver-
age over 80% of the radiopharmaceuticals used in clinical
applications are labeled with 99mTc (T = 6 h), the short-
½
lived, metastable nuclide having nuclear properties that
are almost optimal with regard to a low radiation expo-
sure of the patient.
In an effort to obtain a precursor that can localize
and image cancer cells in vivo and to pretarget drugs to
tumors, 99mTc labeled DOTA-Triazole-Biotin has been
synthesized by conjugating DO3A-functionalized alkyne
with biotinylated azide using click chemistry and evalu-
ated for targeted applications.
Materials and methods
2-Bromoethylamine hydrobromide, Sodium azide, Bio-
tin, N-hydroxysuccinimide, Dicyclohexylcarbodimide,
Copper sulfate, Sodium ascorbate, Propargyl bromide,
and tert-butylbromoacetate were purchased from Sigma-
Aldrich and Merck. All solvents used were of analytical
grades. For reactions to be performed under dry condi-
tions, solvents were dried by usual reported laboratory
procedures. 1, 4, 7, 10-tetraazacyclododecane-1, 4, 7-tris
(t-butyl acetate) was synthesized in our laboratory us-
ing the method of Li and Wong (2002). TLC was run on
the silica gel-coated aluminum sheets (Silica gel 60 F254
,
Another advantage of using click chemistry is that it
tolerates most of the organic functional groups, proceeds
in a variety of solvents, and performs well over a broad
pH and temperature range (Lutz & Zarafshani, 2008).
e 100% atom economy and simple product isolation
make this reaction useful in various applications ranging
from bioorthogonal bioconjugation, polyaminocarboxy-
lic acids synthesis to the construction of peptide bond
surrogates (Vundyala et al., 2008). ere are important
clues that suggest that the triazole group displays struc-
tural similarity with the amide bond. e lone pair of the
3-nitrogen mimics the one of the carbonyl oxygen of the
amide bond; the polarized C(5)-H bond can act as a hy-
drogen bonding donor, just like the amide N–H bond, and
the electrophilic and polarized 4-carbon is electronically
similar to the carbonyl carbon. Since the overall dipolar
Merck, Germany) and visualized in UV light 254nm. Ra-
diocomplexation and radiochemical purity was checked
by instant thin layer chromatography. 99mTc was procured
from Regional Center for Radiopharmaceuticals (North-
ern Region), Board of Radiation and Isotope Technology
(BRIT), Department of Atomic Energy, India.
Cell culture
Monolayer cultures of U-87MG (obtained from
NIMHANS, Bangalore), were maintained at 37°C in a
humidified CO2 incubator (5% CO2, 95% air) in DMEM
(Sigma, USA) supplemented with 10% fetal calf serum
(Biological industries, Israel), 50 U/ml penicillin, 50 μg/
ml streptomycin sulfate and 2 μg/ml nystatin. Cells were
routinely subcultured twice a week using 0.05% Trypsin
(Sigma, USA) in 0.02% EDTA.
G© 2011 Informa UK, Ltd.

420 J.K. Uppal et al.
Synthesis of 5-(2-Oxo-hexahydro-thieno [3, 4-d]imidazol-4-
yl)-pentanoic acid (2-azido-ethyl)-amide(4)
Instrumental analysis
1Hand13CNMRspectrawererecordedonaBrukerAvance
II 400 MHz system (Ultra shield). Mass spectra (ESI-MS in
positive and negative ion mode) were performed on in-
house Agilent 6310 system ion trap. e receptor binding
data were analyzed using iterative curve-fitting program
EQUILIBRATE software from graph pad. Radioimaging
was performed using HAWKEYE gamma camera and
single well type capintec γ- scintillation counter respec-
tively.
To a solution of the Biotin N-hydroxysuccinimide ac-
tive ester (500 mg, 1.466 mmol) in dimethylformamide
(10 ml) was added triethylamine (740 mg, 7.33 mmol) at
room temperature. is was followed by the addition of
2-azidoethylamine (252 mg, 2.932 mmol) to the reaction
mixture and the reaction was run for 48 h. e solvent was
evaporated under reduced pressure to obtain the crude
product. It was purified by column chromatography on
a silica gel (eluent: dichloromethane/methanol: 9/1) to
afford the desired product as a white solid (70%). e
final product was well characterized by 1H, 13C NMR and
MS. 1H NMR (DMSO, 400 MHz) δ ppm: 1.21-1.51(m, 6H),
2.19(t, 2H), 2.50(t, 2H), 2.71(s, 4H), 2.83(t, 2H), 3.31(t,
2H); 3.34 (s, 1H), 4.30-4.40(m, 2H), 6.43(brs, NH); 13C
NMR (DMSO, 400 MHz) δ ppm: 25.00, 29.71, 31.19, 33.9,
36.21, 55.85, 59.64, 61.51, 163.16, 173.24; MS (ESI+) m/z:
313[M+H]+
Animal models
Animal protocols have been approved by Institutional
Ethics Committee. Mice were housed under conditions
of controlled temperature 22°C and normal diet. Athymic
mice were inoculated subcutaneously with 0.1 ml cell
suspension (1 × 106 U-87 MG cells) in the right thigh un-
der sterile conditions.
Synthesis
Synthesis of (4, 7-bis-tert-butoxycarbonylmethyl-10-prop-
2-ynyl-1,4,7,10 tetraaza-cyclododec-1-yl)-acetic acid t-butyl
ester (6)
e
ligand
[4,7-bis-carboxymethyl-10-(1-{2-[5-
(2-oxo-hexahydro-thieno[3,4-d]imidazol-6-yl)-
pentanoylamino]-ethyl}-1H-[1,2,3]triazol-4-ylmethyl)-
1,4,7,10 tetraaza-cyclododec-1-yl]-acetic acid was
synthesized as follows (Scheme 1).
To a solution of 1, 4, 7, 10-tetraazacyclododecane-1, 4,
7-tris (t-butyl acetate) (500 mg, 0.9727 mmol) in ace-
tonitrile (50 ml) was added potassium carbonate (1.34 g,
9.7276 mmol) under nitrogen. e reaction temperature
was raised to 70°C followed by the dropwise addition of
propargyl bromide (231 mg, 1.94 mmol). e reaction
was stirred at 70°C for 12 h. e reaction mixture was
cooled to room temperature, filtered, and the filtrate was
evaporated under reduced pressure to obtain the crude
product. It was purified by column chromatography on
a silica gel (eluent: dichloromethane/methanol: 9/1)
to afford the desired product as a brown oil (80%). e
final product was characterized by 1H, 13C NMR, and MS.
1H NMR (CDCl3, 400 MHz) δ ppm: 1.40(s, 27H), 2.70-2
.82(m, 16H), 2.15(s, 1H) 3.31(s, 6H), 3.46(s, 2H); 13C NMR
(CDCl3, 400 MHz) δppm: 29.32, 43.08, 51.55, 51.94, 56.81,
65.82, 80.87, 172.97; MS (ESI+) m/z: 553[M+H]+
Experimental
Synthesis of 2-Azido-ethylamine (2)
To a solution of 2-bromoethylaminehydrobromide
(1 g, 4.87 mmol) in water (10 ml) was added sodium
azide (951 mg, 14.6 mmol) and the reaction tempera-
ture was raised to 80°C. The reaction was run for 18 h
and reaction progress was monitored by TLC. The re-
action mixture was cooled to room temperature and
extracted with diethyl ether (3 × 50 ml). The combined
organic extracts were dried over anhydrous Na2SO4
and evaporated to dryness under reduced pressure to
obtain colorless oil (85%). The final product was well
characterized by 1H, 13C NMR and MS. 1H NMR (CDCl3,
400 MHz) δ ppm: 2.88(t, 2H); 3.39(t, 2H); 13C NMR
(CDCl3, 400 MHz) δ ppm: 41.32; 54.67; MS(ESI+) m/z:
87[M+H]+
Synthesis of [4-tert-Butoxycarbonylmethyl-7-(2-hydroxy-
allyl)-10-(1-{2-[5-(2-oxo-hexahydro-thieno[3,4-d]imidazol-
6-yl)-pentanoylamino]-ethyl}-1H-[1,2,3]triazol-4ylmethyl)-
1,4,7,10 tetraaza-cyclododec-1-yl]-acetic acid tert-butyl ester
(7)
Biotinylated azide (4; 200 mg, 0.6410 mmol) and N-
functionalized DO3A alkyne (5; 354 mg, 0.6410 mmol)
were mixed in 1:1 water/t-butanol (10 ml). is was fol-
lowed by the addition of copper(II) sulfate pentahydrate
(16 mg, 0.0641 mmol) and sodium ascorbate (127 mg,
0.6410 mmol).e reaction was stirred for 24 h at room
temperature. NH4OH was added to the resulting green
solution to trap copper(II). e reaction mixture was
extracted with ethyl acetate (3 × 50 ml) and combined or-
ganic extracts were dried over sodium sulfate, evaporat-
ed under reduced pressure to obtain the crude product.
It was purified by column chromatography on a silica gel
Synthesis of Biotin-N-hydroxysuccinimide ester (3)
To a solution of biotin (1 g, 4.08 mmol) in dimethylfor-
mamide (5 ml) was added dicyclohexylcarbodimide (1 g,
4.89 mmol) at 0°C. N-hydroxysuccinimide (562 mg, 4.89
mmol) was added to the reaction mixture and reaction
was stirred for 24 h. e reaction mixture was filtered and
filtrate was evaporated under reduced pressure to ob-
tain the crude product which was purified by etheration
under cold conditions (60%). e final product was well
characterized by 1H, 13C NMR, and MS.
1H NMR (DMSO, 400 MHz) δ ppm: 1.22-1.65(m,
6H), 2.48(t, 2H), 2.55(t, 2H), 2.63(s, 4H), 3.12(s, 1H),
4.30-4.40(m, 2H), 6.36 (brs, NH); 13C NMR (DMSO,
400 MHz) δ ppm: 24.91, 28.28, 30.44, 31.21, 33.79, 55.68,
59.61, 61.43, 163.12, 170.72; MS (ESI+) m/z: 341[M+H]+
Journal of Drug Targeting

Evaluation of DOTA-Triazole-Biotin based tumor pretargeting 421
Scheme 1(a)
NH2
Sodium Azide
Water
NH2
Br
N3
2
1
O
Biotin-NHS (3)
DMF
NH
HN
NH
S
N3
4
O
Biotinylated azide
Scheme 1(b)
Ot-Bu
O
NH
NH
HN
N
N
N
N
1.t-butylbromoacetate
2.Propargylbromide
t-BuO
O
Ot-Bu
O
HN
5
6
O
S
Biotinylatedazide
Cu(I); Sodium ascorbate
NH
HN
H
N
N
C
O
N
N
Ot-Bu
O
N
N
N
N
t-BuO
O
Ot-Bu
O
7
O
S
NH
HN
Trifluoroacetic Acid
H
N
N
N
C
O
N
OH
O
N
N
N
HO
O
OH
N
O
8
DOTA-Triazole-Biotin
Scheme 1. Synthesis of DOTA-Triazole-Biotin via Cu(I) catalyzed click conjugation.
(eluent: dichloromethane/methanol: 9/1) to afford the
desired product as a white solid (80%). e final product
was well characterized by 1H, 13C NMR and MS. 1H NMR
(CDCl3, 400 MHz) δ ppm: 1.22-1.65(m, 6H), 2.19(t, 2H),
2.46-2.67(m, 16H), 3.12(s, 1H), 3.32(s, 6H), 3.56-3.91(m,
6H), 4.30-4.40(m, 2H), 6.36 (brs, NH), 7.38(s, 1H); 13C
NMR (CDCl3, 400 MHz) δ ppm: 24.95, 28.09, 29.32, 29.99,
31.88, 33.89, 52.30, 53.89, 55.68, 59.61, 61.43, 163.12,
170.72; MS (ESI+) m/z: 866[M+H]+
Synthesis of [4,7-bis-carboxymethyl-10-(1-{2-[5-(2-oxo-
hexahydro-thieno[3,4-d]imidazol-6-yl)-pentanoylamino]-
ethyl}-1H-[1,2,3]triazol-4-ylmethyl)-1,4,7,10tetraaza-
cyclododec-1-yl]-acetic acid (8)
To a solution of 6 in anhydrous dichloromethane was
added trifluoroacetic acid (5 ml) and the reaction was
stirred for 24 h at room temperature. The reaction
progress was monitored by TLC. The solvent was evap-
orated to dryness under reduced pressure to obtain
G© 2011 Informa UK, Ltd.

422 J.K. Uppal et al.
the crude product. The product was washed well with
chloroform (50 ml × 3). Addition of cold diethyl ether
led to precipitation of pure product as pale white solid
Cytotoxicity studies of DOTA-Triazole-Biotin
Viability assay: Trypan blue exclusion assay
Cell suspension of monolayer culture of U-87MG 1 × 106
cells were prepared and 1:1 dilution of the suspension
using a 0.4% trypan blue solution was done after 2 h treat-
ment with the compound. 10 μl of cell suspension was
loaded on a hemocytometer. e number of stained cells
and total number of cells were counted and percentage
viability was calculated.
1
(60%). The final product was well characterized by H,
13C NMR, and MS. 1H NMR (D2O, 400 MHz) δ ppm:
1.89-2.32(m, 8H), 2.64 (s, 2H), 3.03 (s, 1H), 3.25-3.66
(m, 16H), 3.35 (m, 6H), 3.66 (s, 2H), 3.77-4.03(m, 4H),
4.5-4.67(m, 2H), 6.70 (s, 1H); 13C NMR (D2O, 400 MHz)
δ ppm: 22.54-31.45, 33.93, 40.38, 49.39, 50.29, 129.72,
157.23, 166.85, 173.26; MS (ESI+) m/z: 695.5[M]+
(Figure 1)
Macrocolony assay
Monolayer culture of U-87MG cell line was trypsinized
and 100 to 1000 cells were plated depending upon the
concentrations of the compound in 60-mm petri dishes
and incubated at 37°C in 5% CO2 humidified atmosphere
for 8 days. Colonies were fixed in methanol and stained
with 1% crystal violet. More than 50 colonies were count-
ed.
Synthesis of radiolabeled DOTA-Triazole-Biotin
DOTA-TB (1 mg) was dissolved in 1 ml double distilled
water under nitrogen atmosphere. 100 μl of this solu-
tion was taken in glass vial and stannous chloride (50
μl; 1 mg dissolved in 1 ml 10% acetic acid) was added to
it followed by the addition of 74 MBq of freshly eluted
(<1 h old) 99mTechnetium pertechnetate saline solution.
e pH of the reaction mixture was adjusted to 7 using
0.1 M sodium carbonate solution. e contents of the
vial were mixed well. e vial was allowed to stand for
20 min at room temperature. Standard safety proce-
dures were employed during radiocomplexation of the
chelate.
MTT assay
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 microtiter plate at a uniform cell density of
4000 cells/well 24 h before treatment. Cells were treated
with the varying concentrations of the agent for various
time intervals 24 h, 48 h, and 72 h and MTT assays were
performed. At the end of treatment, negative control and
treated cells were incubated with MTT at a final concen-
tration of 0.05 mg/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
was measured on 150 µl of extracts at 570 nm (reference
filter: 630 nm). Mitochondrial activity 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 nm–630 nm) nega-
tive control] × 100%. Viability at (0.0001–10 mM) concen-
tration range was plotted against time for DOTA-Triazole-
Biotin (Figure 2).
Radiochemical purity of 99mTc-DOTA-Triazole-Biotin
conjugate
e radiolabeling efficiency was determined using as-
cending instant thin layer chromatography on ITLC-SG
(Paul German, USA) using 100% acetone, ternary mix-
ture of pyridine: acetic acid: water (3:5:1.5) and 0.9%
saline as the mobile phase. Each TLC was cut in 0.5 cm
fragments and counts of each segment were taken.
Hence the percentage of free Na99mTcO4 , reduced 99mTc,
−
and complexed 99mTc could be calculated. 99mTc DOTA-
Triazole-Biotin Conjugate remained at the origin while
uncomplexed 99mTc traveled with the solvent front in
acetone.
+MS. 0.0–0.6min #(1–44)
100
90
80
70
60
50
40
30
20
10
0
6
695.5
4
2
864.4
806.4
956.3
0
2
4
6
8
10
750.3
639.4
600
700
Concentration in mM
0
800
900
300
400
500
m/z
200
Figure 2. Colorimetric estimation of the mitochondrial activity
for cytotoxicity of DOTA-Triazole-Biotin (MTT assay) in U-87MG
cell line (0.0001–10mM concentration range).
Figure 1. A representative mass spectrum showing the molecular
ion peak of DOTA-Triazole-Biotin. MS (ESI+) m/z: 695.5[M]+.
Journal of Drug Targeting

Evaluation of DOTA-Triazole-Biotin based tumor pretargeting 423
percentage injected dose per gram of the tissue (%ID/g)
(Table 1).
Cell uptake assay
Cell uptake studies were performed using radiolabeled
DOTA-Triazole-Biotin and compared with avidin-
treated cells for 2 h. e specificity of biotin conjugates
to bind to cell surface receptors on tumor cells was
examined by receptor binding assays on U-87MG cell
line grown in normal DMEM (10% serum). Monolayer
cultures of the cell lines were washed with HBSS and
were then incubated for 2 h in HBSS at 37°C prior to
the experiment. Binding experiments were conducted
at 37°C. e cell line cultures were then incubated
for 40 min with 99mTc-DOTA-Triazole-Biotin at 37°C in
HBSS containing various concentrations (0.001 μM–10
μM) in the absence and presence of the 100 folds ex-
cess unlabeled biotin 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 cold PBS and 0.9% saline four times.
e cell-associated radioactivity was determined by
gamma scintillation counting. Scatchard plot analysis
was done using EQUILIBRATE software from graph pad
(Figure 3).
Results
In a two step pretargeting strategy, binding affinity of avi-
din and biotin have been utilized for tumor targeting. e
10
Control With Avidin
1
0.1
0.01
1.00E-15
1.00E-13
1.00E-11
BOUND
1.00E-09
1.00E-07
Figure 3. Scatchard Plot of the specific binding data to the ratio of
bound to free (B/F) for U-87MG cell line with and without avidin
treated cells.
Blood kinetics
300 μl of the complex 99mTc-DOTA-Triazole-Biotin
(9.8 MBq activity) was injected intravenously through the
dorsal ear vein in normal rabbit. Blood was withdrawn
from the other ear vein at different time intervals (5 min
to 24 h). Persistence of activity in the circulation was cal-
culated, assuming total blood volume as 7% of the body
weight (Figure 4).
50
40
t1/2 = 75 min
t
1/2 = 8h 11 minutes
30
20
10
0
Avidin pretargeting scintigraphy
Tumor imaging was performed in U-87 cell line implant-
ed tumor bearing nude mice (n= 5) following administra-
tion of 100 µl of the labeled conjugate 8 (40 μg, 3.7 MBq
activity) pretreated with avidin for 2 h prior to injection.
Mice were coinjected with unlabeled biotin to estimate
the displacement with respect to radiolabeled biotin-
DOTA conjugate. Images were obtained using HAWK-
EYE gamma camera, at different time intervals (15 min
to 24 h) postinjection (Figure 5). Semiquantative analysis
by generation of region of interest (ROI) was done using
INTEGRA software to evaluate the uptake of the admin-
istered compound at different time intervals.
0
5
10
15
20
25
Time (in hour)
Figure 4. Blood clearance of 99mTc-DOTA-Triazole-Biotin (9.8
MBq activity) administered through ear vein in normal rabbit.
2h block
Tumor
Biodistribution
U-87 cells were injected subcutaneously in the thigh of
the right hind leg of nude mice. When the tumors became
conspicuous and approximately of 0.17 g, the mice were
used for biodistribution study. An intravenous injection
of 99mTc DOTA-Triazole-Biotin conjugate (100 μl; 3.7 MBq
activity) was injected through the tail vein of each mice.
Mice were dissected at 1 h, 4 h, and 24 h postinjection;
different tissues were isolated, weighed and counted in a
gamma counter calibrated for 99mTc energy. Uptake of the
radiotracer in each tissue was calculated and expressed as
Figure 5. Whole body γ image of female athymic mice with U-87
tumor in right thigh at 1h following intravenous administration of
99mTcDOTA-Triazole-Biotin(2.96MBqactivity).Radiotraceruptake
can be visualized in tumor with and without coadministration of
blocking dose (cold biotin).
G© 2011 Informa UK, Ltd.

424 J.K. Uppal et al.
Table 1. Biodistribution of 99mTc-DOTA-Triazole-Biotin in
athymic mice following intravenous injection.
rocolony assay. Analyzing the MTT assay data at a range
of 0.1 µM to 10 mM concentrations, it was observed that
at a concentration of 10 mM of compound after 24 h ex-
posure resulted in 35% reduction in the MTT assay which
indicated 35% cell death. Whereas at lower concentra-
tions the conjugate did not show any adverse effect on
the cell survival as only 5% of the cell death was observed
when 1 μM of the DOTA-Triazole-Biotin was incubated
with U-87 cell line for 2h. (Figure 2). Time dependent
curve showed regain of the metabolic viability of U87-
MG cells at 72 h post treatment.
Cell uptake studies: e ability of DOTA-Triazole-
Biotin to bind to U-87 tumor cell lines pretreated with
0.1 mg/ml of avidin was determined by saturation bind-
ing assay using 99mTc-DOTA-Triazole-Biotin as the labeled
ligand. Nonspecific binding was obtained by using 100
fold excess of unlabeled biotin. Analysis of the binding
curve exhibited saturable binding of the radioconjugate
in nM range. Scatchard plot analysis revealed that the la-
beled compound exhibited high affinity on U-87 cell line
with a Kd value of 69 nM. is value was found to be 100
folds more than the Kd (0.37 μM) obtained when the cells
were not treated with avidin (Figure 3).
Blood kinetics: Blood kinetics studies in rabbits depict-
ed that there was a rapid clearance of the conjugate 8 from
the body as only 16% of injected activity persisted in the
circulation at 1h. After 2h, the clearance followed a slow
pattern and at 24h approximately 1.05 % activity remained
in the blood (Figure 4). e biological half-life was ob-
tained to be t1/2 (Fast): 75min; t1/2 (Slow): 8h and 11min.
Scintigraphy: Imaging of animals was performed at
different time intervals following intravenous adminis-
tration of the radiolabeled conjugate in five mice. e
mice illustrated the beginning of accumulation of ac-
tivity in tumor at 30 min, which reached to maximum
at 1 h (Figure 5) and remained stable for 4 h. Semiquan-
titative analysis was generated from Region of Interest
(ROI) placed over areas counting average counts per
pixels with maximum radiotracer uptake at the tumor
site and compared to symmetric counterparts with ROI
in soft tissues. Target to nontarget (T/NT) ratio at dif-
ferent time intervals was calculated. e ratio of tumor
to soft tissue at 1 h was found to be 23 2.3 which then
increased to 36 4.2 at 4 h. e radiotracer uptake was
decreased at 24 h but the ratio was increased due to the
clearance of the labeled compound from the body. e
T/NT ratio obtained was 20.8 3.66 at 24 h in tumor
bearing nude mice.
Biodistribution: Athymic mice implanted with U-87
cell line exhibited major accumulation in kidneys
(7.5%/g) followed by liver (2.7%/g) at 2 h showing that
the complex is excreted both by renal as well as hepa-
tobiliary routes (Table 1). e high tumor accumulation
of 13.30 0.08 % ID/g at 2 h that remains high 21.4 1.07
up to 4 h post injection was observed. is uptake was
significantly blocked by (> 90%) in the animals receiv-
ing a high dose of avidin together with the radiolabeled
compound 8. As the background activity gradually
Organs
Blood
%ID/ga (2h)
1.32 0.22
0.23 0.06
1.52 0.13
2.7 0.27
%ID/ga (4h)
0.93 0.16
0.21 0.05
1.46 0.11
2.63 0.28
0.36 0.09
6.3 0.7
%ID/ga (24h)
0.24 0.05
0.08 0.01
0.47 0.09
1.65 0.31
0.22 0.08
1.53 0.22
0.25 0.04
0.17 0.06
0.11 0.03
1.23 0.24
11.18 1.64
Heart
Lungs
Liver
Spleen
Kidneys
Stomach
Intestines
Muscles
Tumor
0.47 0.12
7.5 0.68
0.34 0.12
0.51 0.17
0.23 0.04
3.06 0.75
0.3 0.05
0.38 0.08
0.17 0.06
3.65 0.97
21.4 1.07
Tumor/Muscle 13.30 2.08
aPercentage of injected 99mTc-DOTA-Triazole-Biotin dose per gram
following intravenous administration, values shown represent the
b
mean standard deviation of data from three animals. Tumor/
background ratios based on corresponding % injected dose per
gram data.
covalent attachment of bifunctional chelating agents to
biotin is a feasible process as biotin has a free carboxylate
group and this functional group is not involved in bind-
ing of biotin to avidin. e carboxylate group of the biotin
was activated by formation of N-hydroxysuccinimide
(NHS) ester. For the synthesis of biotin functionalized
azide, 2-bromoethylamine was reacted with sodium az-
ide at 80°C followed by its coupling with the biotin-NHS
active ester at room temperature. e propynyl derivative
was prepared by the alkylation of trisubstitued cyclen
with propargyl bromide in presence of potassium car-
bonate at 70°C. e key step in the synthesis was Cu(I)
catalyzed 1, 3-dipolar cycloaddition of the biotinylated
azide with the propynyl functionalized DO3A affording
1,4-disubstitued triazole in good yields. e final step
involved deprotection of the t-butyl groups with trifluo-
roacetic acid yielding the desired chelate DOTA-Triazole-
Biotin. All intermediates and the final compound were
successfully characterized by spectroscopic techniques
such as 1H, 13C NMR and MS (Figure 1).
Qualitycontrolof99mTcDOTA-Triazole-Biotin:Ascend-
ing thin layer chromatography illustrated that labeled
complex remained at the origin while 99mTcO− traveled
4
up with the solvent front in acetone. e yield of the free
and complexed ligand could be calculated from above
observations. e labeling yield was found to be greater
than 98%. Percentage radiolabeling was calculated for
different time intervals, namely, 0, 2, 4, 6 and 24 h. e
radiolabeled complex exhibited sufficient stability up to
24 h with 97% of the radiolabeling efficiency.
Cytotoxicity studies of DOTA-Triazole-Biotin
e percentage of viable cells for 2 h treatment of the title
compound as determined by the trypan blue exclusion
assay was found to be 45% at 50mM. e IC50 value for
the compound was 40 2.7 mM as calculated from the
cell viability assay. U-87 MG showed survival value of
0.88 0.06 at 1 mM for unlabeled compound. Surviving
fraction of 0.72 0.05 at 10 mM was observed in the mac-
Journal of Drug Targeting

Evaluation of DOTA-Triazole-Biotin based tumor pretargeting 425
cleared from the body, there was an increase in target-
to-nontarget ratios obtained (Table 1).
tolerance, and easy access to gram-scale preparation of
regiospecific 1,4-disubstituted 1,2,3-triazoles.
Radiochemical analysis demonstrated that the chelate
formed stable electron donor complexes with 99mTc with
high reproducible labeling efficiency. is compound
was examined for its ability to induce cytotoxicity in the
cancer cell lines using trypan blue dye exclusion assay to
determine the percentage of viable cells. Macrocolony
assay was done to test the ability of cells to form colo-
nies after treatment with the compound. MTT assay was
done to analyze the metabolic activity posttreatment.
Cytotoxicity studies were found to be in good correlation
with the different techniques used to investigate the cy-
totoxicity of the compound. e IC50 value of 40 mM was
found to be well in range as for imaging 0.102 mmol/kg
of the compound is being injected for human scan. e
blood clearance studies of the radiolabeled compound
8 showed its high target uptake with the diagnostically
useful target-to-nontarget ratio in a short period of time.
It exhibited rapid clearance (avidin clearing compound)
from blood circulation thereby resulting in the reduction
of the background activity. e saturation binding assay
revealed that 99mTc-DOTA-Triazole-Biotin has a high af-
finity on U-87MG cell line (pretreated with avidin) with
a Kd value of 69 nM. e high specificity of 8 results in
selective uptake and distribution of the radiolabeled li-
gand that can be visualized in the high quality images,
obtained 1 h after administration in animal models with
and without coadministration of blocking dose (biotin),
which allows the advantage of comparing in vitro recep-
tor binding studies and tumor-bearing animal studies.
e biodistribution studies illustrated that 99mTc-labeled
DOTA-Triazole-Biotin persisted in liver up to 4 h and is
cleared through renal and hepatobiliary routes. Imag-
ing of tumor-bearing nude mice correlated well with the
biodistribution studies. us our preclinical evaluations
and results demonstrate an encouraging and promising
future of this compound in the field of two-step pretar-
geting.
Discussion
Conventional nuclear medicine imaging with radiola-
beled tumor specific agents such as antitumor antibod-
ies can provide high tumor/nontumor ratios but usually
with slow signal localization and clearance. is paper
describes a method for two-step pretargeting of tumors
DOTA-Triazole-Biotin, synthesized through an efficient
click reaction of a biotinylated azide and alkyne function-
alized DOTA nucleus. In order to increase the amount of
radioactivity bound to cancer cells, a new approach in
cancer therapy called pretargeting has been employed.
e pretargeting protocol involved a two-step procedure
comprising prior administration of avidin to establish
secondary binding sites on the tumor that could be more
efficiently targeted by low molecular weight radiolabeled
biotinylated chelate (99mTc DOTA-Triazole-Biotin). is
led to high tumor/nontumor ratios as the radiolabeled
biotin chelate was placed on a small molecule avidin
designed to clear from the circulation and whole body
rapidly eliminating excessive irradiation to nontarget
tissues. e extremely high binding affinity of biotin
to avidin provided a high tumor targeting efficiency of
DOTA-Triazole-Biotin and the tetrameric architecture of
avidin (four fold valency for biotin) offered the potential
of modest signal amplification at the tumor site.
It is observed that a short distance between avidin and
the surface of the biotinylated molecule might result in
unsatisfactory binding of the biotinylated chelate to avi-
din. For sufficient binding capability to avidin, the biotin
site and the chelating site of the biotinylated conjugate
must be separated by a spacer. erefore a dynamic strat-
egy involving click chemistry was used to incorporate a
highly stable triazole spacer between biotin and the
macrocycle as well as to simplify the synthesis of biotin
DOTA conjugate. Triazoles are stable to acid and basic
hydrolysis and reductive and oxidative conditions, in-
dicative of a high aromatic stabilization. Ever since their
debut in 2001, click reactions are finding more and more
applications in organic synthesis. is Cu(I) catalyzed di-
polar cycloaddition owes its usefulness in part to the ease
with which azides and alkynes can be introduced into a
molecule and their relative stability under a variety of
biological and organic conditions. e synthesis of biotin
functionalized azide was a convenient process utilizing
readily available starting materials and simple reaction
conditions. It was thereupon subjected to Cu(I) catalyzed
1,3-dipolar cycloaddition with its alkyne partner, namely,
propynyl derivatized DO3A leading to high yield synthe-
sis of DOTA-Triazole-Biotin thereby eliminating the ex-
tensive protection and deprotection steps encountered
in the routine synthesis of DOTA-biotin conjugates. Click
conjugation offered numerous advantages of overall good
yields, benign reaction conditions, high functional group
Conclusion
To summarize, a new candidate for avidin based two-step
pretargeting of tumors has been synthesized and evalu-
ated for potential imaging and diagnostic applications.
An expedient methodology to derivatize macrocyclic
chelating agents with biotin using click chemistry lead-
ing to the synthesis of DOTA-Triazole-Biotin has been re-
ported. e chelate possesses high stability in biological
environment and exhibits effective interaction with its
avidin target and low nonspecific retention in vivo.
Acknowledgements
We are grateful to Dr. R. P. Tripathi, Institute of Nuclear
Medicine and Allied Sciences, University of Delhi, for
providing excellent research facilities.
G© 2011 Informa UK, Ltd.

426 J.K. Uppal et al.
molecule for pretargeted radionuclide therapy. Bioconjug Chem,
16, 1468–1474.
Declaration of Interest
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