Synthesis, biological evaluation and molecular docking studies of high-affinity bone targeting N,N'-Bis (alendronate) diethylenetriamene-N,N'-triacetic acid: A bifunctional bone scintigraphy agent

Article abstract of DOI:10.1111/cbdd.12194

A bisphosphonate derivative DTPA-bis(alendronate) conjugate has been synthesized and evaluated as potential radiopharmaceutical for bone imaging. The compound was synthesized by the covalent coupling of DTPA-bis(anhydride) with alendronate and was char-acterized on the basis of IR, NMR and mass spectroscopy. It was labelled with ^99mTc with 96% efficacy and was found stable for about 24 h under physiological conditions. Blood kinetic studies of ^99mTc DTPA-bis(alendronate) showed a biexponential pattern as well as quick washout from the blood circulation. The biological t_1/2(F) and t_1/2(S) were found to be 50 min ± 0.001 and 6 h 30 min ± 0.005, respectively. Imaging and biodistribution studies showed a significant accumulation of ^99mTc DTPA-bis(alendronate) conjugate at bone site. Bone-to-muscles ratios were 12.08 ± 0.001 at 1 h, 45.33 ± 0.001 at 4 h and 35.83 ± 0.001 at 24 h after post-injection, respectively. The receptor binding of the ^99mTc-DTPA-bis (alendronate) was established on human bone cell line (Soas-2) revealed K_D = 0.86 nm. The preliminary result of the ^99mTc-DTPA-bis(alendronate) is encouraging to carrying out further in vivo experiment for targeted bone imaging because of good-bone-to-normal-organ contrast. Further docking analysis with molecular targets, farnesyl diphosphate synthase, geranylgeranyl pyrophosphate and osteocalcin revealed the high affinity of -17.419 and thus represents strong potential of bone-imaging agent.

Full text of DOI:10.1111/cbdd.12194

Chem Biol Drug Des 2013; 82: 468–476
Research Letter
Synthesis, Biological Evaluation and Molecular
Docking Studies of High-Affinity Bone Targeting
N,N -Bis (alendronate) Diethylenetriamene-N,N’-Triacetic
’
Acid: A Bifunctional Bone Scintigraphy Agent
1
,2
1
1
Nidhi Chadha , Deepa Sinha , Anjani K. Tiwari ,
Bone metastasis is a major complication of several differ-
ent cancers, may be the first indication that the disease
has spread beyond the local area and that the prognosis
may have worsened. In recent years, several molecules
have been identified as new therapeutic agents for the
diagnosis and therapy of bone disease and of these the
most important is bisphosphonate (1), which are taken up
by bone, bind to the bone mineral surface and inhibit
osteoclastic bone resorption. For the purpose of bone
scanning, the most frequently used phosphonate ana-
logues were methylene diphosphonate (MDP) and ethylen-
ediaminetetramethylene phosphonate (EDTMP) (2,3).
1
1,
Krishna Chuttani and Anil K. Mishra *
1
Division of Cyclotron and Radiopharmaceutical Sciences,
Institute of Nuclear Medicine and Allied Sciences, Brig. S.
K. Mazumdar Road, Timarpur, Delhi 110054, India
Department of Chemistry, University of Delhi, Delhi
10007, India
Corresponding author: Anil K. Mishra,
akmishra63@gmail.com
2
1
*
A
bisphosphonate derivative DTPA-bis(alendronate)
conjugate has been synthesized and evaluated as
potential radiopharmaceutical for bone imaging. The
compound was synthesized by the covalent coupling
of DTPA-bis(anhydride) with alendronate and was char-
acterized on the basis of IR, NMR and mass spectros-
The bisphosphonates that inhibit calcium phosphate crys-
tal dissolution in vitro led to hypothesize that these com-
pounds might also act on bone resorption in vivo (4–6).
The decrease in resorption is accompanied by an increase
in both the calcium balance and the mineral content of the
bone. Bisphosphonates prevent bone loss in many experi-
mental diseases (7–9) and have direct effects on the
tumour cells themselves, they have been shown to
decrease the proliferation and viability of human tumour
cells lines, as well as to increase the apoptotic index of
the human tumour cell lines (10). There is now evidence
that bisphosphonates can decrease multiplication of
tumour cells in vitro and can also induce their apoptosis
by inhibiting the mevalonate pathway (1).
9
9m
copy. It was labelled with
was found stable for about 24 h under physiological
Tc with 96% efficacy and
9
9m
conditions. Blood kinetic studies of
alendronate) showed a biexponential pattern as well
as quick washout from the blood circulation. The bio-
logical 1/2(F) and 1/2(S) were found to be
0 min Æ 0.001 and 6 h 30 min Æ 0.005, respectively.
Imaging and biodistribution studies showed a signifi-
Tc DTPA-bis
(
t
t
5
9
9m
cant accumulation of
conjugate at bone site. Bone-to-muscles ratios were
Tc DTPA-bis(alendronate)
1
2.08 Æ 0.001 at 1 h, 45.33 Æ 0.001 at 4 h and
5.83 Æ 0.001 at 24 h after post-injection, respectively.
3
9
9m
The receptor binding of the
Tc-DTPA-bis (alendro-
nate) was established on human bone cell line (Soas-2)
revealed K = 0.86 nM. The preliminary result of the
Tc-DTPA-bis(alendronate) is encouraging to carry-
Based on these assumption, multidentate phosphonate
chelate having DTPA backbone was synthesized. In the
D
9
9m
99m
99m
case of
Tc-MDP and
Tc-HMDP, the phosphonate
ing out further in vivo experiment for targeted bone
imaging because of good-bone-to-normal-organ con-
trast. Further docking analysis with molecular targets,
farnesyl diphosphate synthase, geranylgeranyl pyro-
phosphate and osteocalcin revealed the high affinity of
À17.419 and thus represents strong potential of bone-
imaging agent.
groups co-ordinate with technetium, which might decrease
the inherent accumulation of MDP and HMDP in bone. In
99m
99m
clinical practice,
Tc-methylene diphosphonate ( Tc-
MDP) is widely used for imaging but has no significant
therapeutic applications. Further, it suffers from suboptimal
properties due to poorly defined chemical species distribu-
tion and the same diphosphonate group responsible for
9
9m
both complexation with
Tc and uptake in the skeleton.
Key words: alendronate, bone imaging, docking, radiophar-
maceutical, spectroscopy
99m
Tc-MDP bone scans require a waiting period of 2–4 h
after injection for complete washout from soft tissue
to occur. Thus, we hypothesized that bone affinity of
Received 18 March 2013, revised 14 June 2013 and
accepted for publication 11 July 2013
99m
Tc-labelled bisphosphonate would be increased by the
bisphosphonate in which the phosphonate groups do not
4
68
ª 2013 John Wiley & Sons A/S. doi: 10.1111/cbdd.12194

SPECT Agent for Bone Imaging
co-ordinate with technetium, and we attempted to develop
-CO-NH-), 3.67 (s, 8H, -CH -COOH), 3.276 (t, 4H, -CH
NH ), 3.218 (s, 2H, CH -COOH), 2.8 (s, 8H, N-CH -CH -N),
2 2 2
2.6 (s, -OH), 1.8 (t, 8H, -CH -CH -). C NMR (D O, ppm):-
2
2
-
9
9m
a
Tc-chelate–conjugated bisphosphonate based on the
2
2
2
2
13
concept of bifunctional radiopharmaceuticals. In this study,
DTPA was chosen as chelating sites, as it has been widely
178.49 (-COOH), 171.01 (-CO-NH-), 74.82, 57.03, 56.19,
9
9m
used for
Tc labelling of proteins, peptides and macro-
52.70, 49.26, 40.01, 30.39, 22.45. EI-MS: m/z calcd 889,
+
molecules. DTPA was conjugated to 4-amino-1-hydroxy-
butylidene-1, 1-bisphosphonate (alendronate), which is a
bisphosphonate analogue and a potent inhibitor of bone
resorption that has been extensively studied in the treat-
ment of postmenopausal osteoporosis and in the preven-
tion of postmenopausal bone loss. The potential of N,
found [M+H O] 917(Figures S1 and S2).
2
Radiolabelling of the DTPA-bis(alendronate) with
9
9m
Tc
Radiolabelling of the DTPA-bis(alendronate) was carried
out by following the procedure described in the literature
(12,13). It was performed by taking 100 lL of 3 mg solu-
’
N -bis (alendronate)diethylenetriamene-N,N’-triacetic acid,
abbreviated as DTPA-bis (alendronate) as a bone-specific
radiopharmaceutical was evaluated by understanding its
biodistribution pattern in mice, its imaging study in normal
rabbit and its receptor-binding assay on human bone cell
tion of the compounds dissolved in water and taken in a
À2
shielded vial. Further 60 lL of 1 9 10 M SnCl
2
. 2H
2
O
(dissolved in N -purged 1 mL 10% acetic acid) was added
2
99m
99m
À
line after its synthesis and radiolabelling with
Tc. Also,
followed by freshly eluted saline solution of
(45 MBq, 100 mL). The pH of the reaction mixture was
adjusted to 6.5 with 0.1 M NaHCO solution. The vial was
TcO
4
some aspects of its binding with its molecular targets were
studied by computational analysis.
3
allowed to incubate for 20–30 min at room temperature.
Labelling of the compounds, radiochemical purity as well
99m
Materials and Methods
as R
f
of the
Tc-based complex was determined by
ITLC-SG strips using 0.9% NaCl aqueous solution (saline)
as developing solvent and simultaneously in acetone and
pyridine, acetic acid and water in 3:5:1.5 ratio (PAW). Each
ITLC was cut into 0.1 cm segments and counts of each
segment were taken.
Experimental section
All chemicals used in the present study were of analytical
grade and purchased from Sigma-Aldrich Co (St Louis,
MO, USA). All the solvents were used after distillation. TLC
was run on the silica gel-coated aluminium sheets (silica
gel 60 F254, E. Merck, KGaA, Darmstadt, Germany) and
visualized under UV light. FTIR spectra were recorded on
the FT-IR Perking Elmer Spectrum BX Spectrophotometer
Biological evaluation
with KBr disks. NMR spectra were recorded in D O on
In vitro serum stability assay
2
Bruker 400 MHz system. ESI spectra were recorded on
The fresh human serum was obtained by allowing blood
collected from healthy volunteers to clot for 1 h at 37 °C
in a humidified incubator maintained at 5% carbon dioxide
and 95% air. Then, the sample was centrifuged at 17.88
9 g, and the serum was filtered through 0.22-lm syringe
filter into sterile plastic culture tubes. The above freshly
6310 Agilent Germany system with ion trap detection with
positive and negative mode. Radio complexation and radio
chemical purity were checked by instant thin layer chro-
matography (silica gel-impregnated paper chromatography)
with ITLC-SG (Gellman sciences, Ann arbar, MI, USA). The
gamma scintillation counting was done at ECA (Electronic
Corporation of India Ltd., Hyderabad, India) Gamma rays
spectrometer K 2700 B. All the reaction steps were moni-
tored by thin layer chromatography (TLC). Millipore-dou-
bled distilled water is used during whole of the procedure.
99m
prepared
Tc radio complexes were incubated in fresh
human serum at physiological conditions, that is, at 37 °C
at a concentration of 100 nM/mL and then analysed by
ITLC-SG at different time intervals to detect any dissocia-
tion of complex. Percentage of free pertechnetate at a
particular time-point that was estimated using saline and
acetone as mobile phase represented percentage dissoci-
ation of the complex at that particular time-point in serum.
Synthesis of DTPA-bis (alendronate)
DTPA-bis (anhydride) was prepared according to the litera-
ture procedure (11) as mentioned in scheme 1. To a stirring
solution of DTPA-bis(anhydride) (35.7 g, 0.1 mol) in 50 mL
of dry DMF, sodium alendronate (71.5 g, 0.22 mol) was
added. Triethylamine was used to adjust the pH of the
reaction mixture at 8. The reaction mixture was stirred for
additional 12 h at 60 °C temperature under nitrogen atmo-
sphere. To the reaction mixture, 100 mL of diethylether was
added and left to stand for 6 h. After filtration and drying in
vacuum, DTPA-bis(alendronate) was obtained as a light yel-
Cell culture
Monolayer cultures of human bone cell line Soas-2 was
maintained at 37 °C in a humidified CO2 incubator (5%
CO , 95% air) in DMEM (Sigma, USA) supplemented
2
with 10% foetal calf serum (Biological Industries Israel
Beit Haemek Ltd., Kibbutz Beit Haemek, Israel), 50 lg/
mL penicillin, 50 lg/mL streptomycin sulphate and
2 lg/mL nystatin. Cells were routinely subcultured once
a week using 0.05% trypsin (Sigma, USA) in 0.02%
EDTA.
low amorphous solid (yield 80.0%). IR (KBr):1550, 1645,
1
3
300 per cm, HNMR (400 MHz, D O):- 7.76 (brs, 2H,
2
Chem Biol Drug Des 2013; 82: 468–476
469

Chadha et al.
Receptor-binding studies
vein. Samples of muscle, stomach, small intestine, kidney,
liver and bone (femur) were taken and weighed. Samples
of different organs were counted in a well-type gamma
scintillation counter to calculate resident activity in different
organs. Tissue concentrations were calculated and
expressed as percent uptake of injected dose per gram or
per millilitre (% ID/g). Bone-to-blood and bone-to-muscle
uptake ratios were determined from the% ID/g values for
the organs. Total volume of the blood was calculated as
7% of the body weight.
6
Exponentially growing cells 0.1 9 10 cells/PD (Soas-2)
were plated at a uniform cell density and incubated over-
night. Monolayer culture (Soas-2) of the cell lines was
washed twice for 2 min with ice-cold binding buffer
(25 mM HEPES, 10 mM MgCl and 1% BSA). The cell line
2
culture were then incubated for 40 min with labelled
compounds (1–8 nM) in the absence and presence of the
1
00-folds excess unlabelled compounds for estimation of
total binding and non-specific binding, respectively. Spe-
cific binding was obtained by subtracting non-specific
binding from total binding. At the end of each experi-
ment, the cells were washed with ice-cold binding buffer
three times for 3 min. The cells were lysed with 200 lL
of lysis buffer. The cell-associated radioactivity was deter-
mined by gamma scintillation counting. Scatchard plot
analysis was done using EQUILIBRATE software from
graph pad (GraphPad Software, Inc., La Jolla, CA, USA).
Docking methodology
For the present study, X-ray crystallographic co-ordinates
of crystal structure of all the targets were obtained from
ꢀ
RCSB PDB; osteocalcin (1QH8) having resolution of 2 A,
molecular targets farnesyl diphosphate synthase (FPPS)
ꢀ
(1UBW, 2.5 A resolution, geranylgeranyl pyrophosphate
ꢀ
(
GPP) (2Z4W, 2.450 A resolution).
Animal models
Docking analysis was performed with glide after preparing
the protein structures with preparation wizard of maestro
(14). During pre-processing, bond orders were assigned;
hydrogen was added to all atoms in the protein structure.
Animal protocols have been approved by Institutional Ani-
mal ethics Committee. Albino New Zealand Rabbits 2–3
Kg and BALB/c mice (22–28 g) were used for blood clear-
ance, and imaging and biodistribution studies. Rabbits
were housed under conditions of controlled temperature of
ꢀ
Water molecules beyond 5 A from hetero (het) groups
were deleted, and missing loops were filled up. States
were generated for these het atoms at pH = 7.0 Æ 3.0. In
the presence of force field OPLS2005, hydrogen atoms of
protein structure were minimized, orientations of retained
water molecules were sampled and pKa’s were deter-
mined. Grid calculations were performed for the protein
active site. No constraints were applied, and rotations of
rotatable groups were disallowed.
2
2 Æ 2 °C and normal diet.
Blood kinetic studies
The blood clearance study was performed in normal rab-
bit, weighing 2–2.5 kg. 18.5 MBq of the
compounds (0.3 mL) was administered intravenously
through the dorsal ear vein. At different time intervals,
about 0.5-mL blood samples were withdrawn from the
dorsal vein of other ear and radioactivity was measured in
the gamma-counter. The data from the experiment were
expressed as percentage of administered dose at each
time interval.
99m
Tc-labelled
The ligands were prepared using the Ligprep
v2.6 module (14). All the possible protonation states at
pH = 7.0 Æ 2.0 were generated using ionization tool.
Specified chiralities of these ligands were retained while
ligand preparation. Low energy conformers were obtained
in the force field OPLS2005 without any constraints.
These conformers were retained for docking studies. Five
thousand poses were kept in the initial phase of the
docking keeping the default scoring window cut-off value
at 100. Ligand Vander Waals radii were scaled to a
factor of 0.80 (default value) for non-polar atoms with a
partial charge cut-off level of 0.15 (absolute value). Post-
docking minimizations were performed by including five
poses per ligand.
Bone scintigraphy
Bone imaging was performed in rabbit by administering
1
00 lL of the labelled conjugate (100 lg, 100 lci). Images
were taken using planar gamma camera equipped with
pinhole collimator. Images were obtained at different time
intervals starting from 15 min to 24 h after post-injection.
Biodistribution study in mice
Glide v5.8 (14) was used for docking analysis. Glide’s
workflow predicts the binding mode of the ligand with high
accuracy. Glide docking uses a series of hierarchical filters
to find the best possible ligand binding locations in a pre-
built receptor grid space that represents the shape and
properties of receptors. It evaluates the energy interactions
of the ligand with the protein in terms of Gscore value that
is used for predicting binding affinity.
BALB/c mice, each weighing 2–3 g, were used to deter-
9
9m
mine the organ distribution of
Tc-DTPA-bis (alendro-
nate). Animal handling and experimentation was carried
out as per the guidelines of the Institutional Animal Ethics
Committee. The mice were killed at 1, 4 and 24 h (six rats
at each time-point) after injection of 18.5 MBq
DTPA-bis (alendronate) in a volume of 0.1 mL via the tail
99m
Tc-
4
70
Chem Biol Drug Des 2013; 82: 468–476

SPECT Agent for Bone Imaging
Results and Discussion
while another peak at 171 ppm corresponding to the
carbonyl group of amide bond, which confirms the
formation of the final conjugate.
Radionuclide bone scanning is an extremely sensitive
method for the detection of various bone diseases and is
considered the most practical screening technique for
assessing skeletal metastases in the whole body. To
achieve our objective of developing new bone-specific
radiopharmaceuticals with enhanced efficiency, a synthetic
strategy was developed wherein two alendronate units
were conjugated with a chelating agent and its bone
uptake properties were compared with other known bis-
phosphonates. Here, diethylenetriaminepentaacetic acid
Radiolabelling of the complex
DTPA-bis(alendronate) was labelled with
99m
Tc using
SnCl .2H O as reducing agent as reported in the literature
2
2
(12,13). All the labelling parameters such as pH, concen-
tration of reducing agents, temperature, etc. were stan-
dardized to achieve the maximum labelling efficiency. The
radiochemical analysis showed that DTPA-bis(alendronate)
99m
99m
(DTPA) was chosen as a
Tc-chelating group because it
is co-ordinated to
Tc radionuclide with more than 96%
99m
forms a relatively stable and hydrophilic complex with
yield. In vitro and in vivo stability of the
Tc-DTPA-bis
9
9m
Tc in high radiochemical yield and purity. Conjugation
(alendronate) was checked in PBS buffer at various time
intervals (1, 2, 4, 6 and 24 h), and the complex was found
to be stable for 24 h with 94% radiolabelling efficiency.
of DTPA-like ligands to macromolecules (peptide, proteins
or bile acids) has been recently pursued to achieve
improved organ specificity. The most popular way to con-
jugate DTPA to other molecules involves the conversion of
two carboxylic groups into a carboxamide via DTPA-bis
In vitro cellular uptake assay
In vitro cellular uptake assay of
99m
(anhydride) (Schemes 1). Besides being selective, due to
Tc-DTPA-bis(alendro-
9
9m
the conjugation with the target-specific molecules, this
methodology has the advantage of convenient synthesis
procedure with high yield. DTPA-bis(alendronate) was syn-
thesized with 80% yield by reacting DTPA-bis (anhydride)
and sodium alendronate in the presence of triethylamine in
DMF. The compound was characterized by spectroscopic
techniques (IR, NMR and mass spectroscopy). One peak
at 3300/cm in FT-IR spectrum corresponds to the mN-H
stretching, and two peaks at 1550 and 1645 per cm are
because of the mN-H and, mC=O stretching, thereby indi-
nate) was performed on bone cell line (Soas-2).
Tc-
Tc-alendronate were taken as controls. Both
99m
DTPA and
99m
99m
Tc-DTPA-bis(alendronate) and
Tc-alendronate had
significantly increased radioactivity localized in the cells
99m
when compared with
tion, maximum uptake of
activity was observed in the cells in comparison with
Tc-DTPA. At 2.5 h after incuba-
99m
Tc-DTPA-alendronate radio-
99m
Tc-DTPA.
1
cated the formation of the amide bond. In the H NMR, a
Receptor-binding studies
singlet at 8 ppm corresponds to the amide proton, which
was further confirmed by C NMR. In C NMR, one peak
at 178.49 confirmed the presence of carboxylic acid group
Scatchard plot analysis shows a good binding affinity of
13
13
the compound towards bone cell lines (Soas-2). K
was found to be 0.86 nM (Figure 1).
D
value
O
O
O
O
HOOC
COOH
Pyridine
N
N
N
O
N
N
N
HOOC
COOH
Acetic anhydride
o
COOH
60 C, 24 h stirring
O
COOH
DMF
NH2
1
2 h stirring
HO
OH
P O
HOOC
COOH
O P
HO OH ONa
N
N
N
N
H
O
COOH
HO
HN
OH
O
NaHO3P
PO H
3
2
Scheme 1: Sythetic scheme of
DTPA-bis (alendronate).
PO HNa
3
PO H
3
2
Chem Biol Drug Des 2013; 82: 468–476
471

Chadha et al.
Figure 1: Scatchard plot of the specific binding data to the ratio
of bound to free (B/F) for Soas-2.
Blood kinetics study
DTPA-bis(alendronate) conjugate shows a very rapid clear-
ance of radioactivity from the blood circulation. Approxi-
mately 78–80% of activity was cleared within 1 h and
more than 90% in 4 h, perhaps due to the specific as well
as hydrophilic nature of the radiotracers, which are
responsible for their fast kinetics (Figure 2), the biological
half-life was found to be t1/2 (F) 50 min Æ 0.01 and t1/2 (S)
Figure 3: Whole body c-scintigraphy of rabbit after 1 h following
9
9m
intravenous injection of
Tc-DTPA-bis(alendronate).
same age and weight demonstrates comparable quality of
99m
99m
image at 3 h with
Tc-MDP and at 1 h with
Tc-
6
h 30 min Æ 0.05, respectively, which is comparable with
99m 99m
DTPA-bis(alendronate). It was observed that the uptake of
the blood clearance of
nate.
Tc-MDP and
Tc-alendro-
99m
the conjugate was faster than that of the
nate and
Tc-alendro-
99m
Tc-MDP alone. Thus, it is concluded that the
synthesized conjugate was a better bone-imaging agent.
Scintigraphy in rabbit
9
9m
Localization of
Tc-DTPA-bis(alendronate) in the rabbit
Biodistribution studies
Biodistribution study of the
over the time was determined by the gamma camera.
Images showed a rapid accumulation of radioactivity in
bone. Imaging of animals was carried out at different time
intervals after administering the labelled compound intrave-
nously through the dorsal ear vein. Within 30 min, appre-
ciable amount of activity was started accumulating in the
joints region. After 1 h, conjugate was localized in the
whole skeleton of the rabbit (Figure 3). Accumulation at
the head region was very prominent. A comparison study
99m
Tc- DTPA-bis(alendronate)
in mice showed remarkable localization at bone (Figure 4).
Very high uptake is seen in early time-points, which
reached a maximum at 1 h (8.82 Æ 0.83% ID/g) and
significant even after 24 h (4.3 Æ 0.44% ID/g). The excre-
tion of the DTPA-bis(alendronate) conjugate is through
renal; therefore, the accumulation in the kidneys showed
high uptake (5.7 Æ 0.42% ID/g) initially at 1 h, but with the
99m
99m
conducted for the
Tc- DTPA-bis(alendronate),
Tc-
9
9m
(alendronate) and
Tc-MDP in the normal rabbit of the
9
9m
Figure 4: Graphical presentation of biodistribution of
Tc-
9
9m
Figure 2: Blood clearance study of
Tc-DTPA-bis(alendronate)
DTPA-bis(alendronate) in rabbit. Data from groups of 3 mice are
in normal rabbit.
expressed as% ID/g Æ SD.
4
72
Chem Biol Drug Des 2013; 82: 468–476

SPECT Agent for Bone Imaging
period of time, it got cleared from the kidneys
(
1.9 Æ 0.25% ID/g at 24 h). Localization of the radioactivity
in liver, stomach and small intestine was low, less than 1%
ID/g at 1 h. In biodistribution studies, the tracer was noted
to persist in high amount in the skeletal system even at
2
4 h that may be due to the higher stability of complex.
99m
Biodistribution of
Tc-DTPA-bis (alendronate) was also
99m
compared with the biodistribution of
Tc-MDP and
9
9m
Tc-alendronate in mice (Table 1). The skeletal uptake
9
9m
of
Tc-DTPA-bis (alendronate) is higher at 1 h as com-
9
9m
99m
pared with
Tc-MDP and
Tc-alendronate. The bone
9
9m
99m
uptake of
biodistribution was found to be 4.92 Æ 0.15% ID/g and
.88 Æ 0.36% ID/g at 1 h, whereas the bone uptake of
Tc-MDP and
Tc-alendronate in mice from
5
9
9m
Tc-DTPA-bis(alendronate) was 8.82 Æ 0.83% ID/g at
h.
1
Docking analysis
Bisphosphonates are known to bind to the calcium ions in
a multidentate manner and thus accumulate in bone
where they inhibit the activity of bone-destroying osteo-
clasts. In the mevalonate pathway, various enzymes are
the molecular target of bisphosphonates such as farnesyl
diphosphate synthase (FPPS), geranylgeranyl pyrophos-
phate (GGPP) in the cell. Being a bone-imaging agent,
these molecules target hydroxyapatite- and calcium-bind-
ing proteins such as osteocalcin. Bisphosphonates tend to
accumulate in osteoclasts at areas of increased bone
metabolism by inhibiting the farnesyl diphosphate syn-
thase. The labelled bisphosphonates were suggested to
have these limitations because the compounds possess
dual activities: one phosphonate group of the BP molecule
acts as a radionuclide chelator and the other phosphonate
group binds to the target(s).
Using the glide docking module, the accurate pose of
DTPA-bis (alendronate) was obtained with good binding
with targets. In case of osteocalcin, GScore of À7.471
represents binding on the surface of osteocalcin including
+
2
interactions with Ca (shown in Figure 5) due to phos-
phonate groups of the molecules. Usually high affinity for
bone-imaging agents is due to presence of amorphous
calcium phosphate in many bone lesions in high concen-
trations which is also an prerequisite too. The –OH on
the phosphonate groups of the DTPA-bis (alendronate)
are suggestive of the bone matrix affinity by binding with
calcium-binding proteins.
Identification of farnesyl diphosphate synthase (FPPS),
geranylgeranyl pyrophosphate (GGPP) as the molecular
targets within the osteoclast is perhaps the most important
finding. From the molecular docking analysis, 1UBW and
2
Z4W revealed strong binding with Gscore of À15.316
and À17.419, respectively. In case of FPPS (1UBW), a key
enzyme in the mevalonate pathway, ligand was to be
+
2
interacting with Mg and positively charged residues such
as Lys214, Arg74, Arg126 and Arg127 (shown in
Chem Biol Drug Des 2013; 82: 468–476
473

Chadha et al.
Figure 5: 2D and 3D interactions diagrams of DTPA-bis(alendronate) with osteocalcin (1QH8).
Figure 6: 2D and 3D interactions diagrams of DTPA-bis(alendronate) with farnesyl diphosphate synthase (FPPS) (1UBW).
Figure 6). Whole molecule was found to be in the cavity of
hydrophobic and negatively charged residues and thus
showed high affinity of FPPS.
molecule in the cavity of protein. The –OH on the
phosphonate groups of the ligand interacted with positively
charged residues Lys174, Lys238, Arg44 and Arg89 and
thus responsible for high Gscore (shown in Figure 7).
Large number of hydrogen bonding networks of the ligand
within the cavity was also found with residues such as
Similarly in case of GGPP (2Z4W), very strong binding is
+
2
observed due to its chelation with two Mg and water
4
74
Chem Biol Drug Des 2013; 82: 468–476

SPECT Agent for Bone Imaging
Figure 7: 2D and 3D interactions diagrams of DTPA-bis(alendronate) with geranylgeranyl pyrophosphate (GPP) (2Z4W).
Tyr210, Phe43. In summary, docking studies depicted
high Gscore values and molecular interactions within the
active sites of these targets and are responsible for high
affinity of these molecules within the bone.
This is also certified that there is no conflict of interest
between the authors of this manuscript.
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Figure S1. H NMR Spectrum of DTPA-bis(alendronate)
Recorded at 400 MHz.
1
13
Figure S2. C NMR Spectrum of DTPA-bis(alendronate)
Recorded at 400 MHz.
42:1234–1238.
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