68Ga-Labeled bismacrocyclic methylene phosphonate as potential bone seeking PET radiopharmaceutical

Article abstract of DOI:10.1016/j.bioorg.2020.104185

Phosphonates-based agents are well-known bone-seeking radiopharmaceuticals with application in detection and therapy. With higher sensitivity and resolution offered by Positron Emission Tomography (PET), tracers based on this technique are gaining huge attention. ⁶⁸Ga-based generator and radiotracers render independence from the on-site cyclotron. We report the development of ⁶⁸Ga-labeled DOTA-based bismacrocyclic phosphonate derivative, for bone PET imaging. The synthesis and characterization of ⁶⁸Ga- DO3P-AME-DO3P was carried out in > 95% purity. The radiotracer displayed high stability and low binding affinity (<3%) to blood serum. High in vitro binding affinity were observed for synthetic hydroxyapatite, SAOS-2, osteoclast and osteoblast cells. In vivo pharmacokinetics revealed fast washout with biphasic release pattern. The deposition of radiotracer in osseous tissues was high (Bone/Muscle ratio:18), as studied from the biodistribution studies. In vivo PET/CT and biodistribution analyses revealed the ability of ⁶⁸Ga-DO3P-AME-DO3P to target and accumulate in bone, thus displaying its potential as a PET bone imaging agent.

Full text of DOI:10.1016/j.bioorg.2020.104185

Bioorganic Chemistry 104 (2020) 104185
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
6
8
Ga-Labeled bismacrocyclic methylene phosphonate as potential bone
seeking PET radiopharmaceutical
a,b
a
a
a
a,⁎
Kanchan Chauhan , Garima Mann , Ambika Parmar Jaswal , Himanshu Ojha , Anil K. Mishra ,
a,⁎
Anupama Datta
a
b
Institute of Nuclear Medicine & Allied Sciences, DRDO, Brig. SK Mazumdar Marg, Delhi 110054, India
Department of Bionanotechnology, Centro de Nanociencias y Nanotecnología, Universidad Nacional Autónoma de México, Km. 107 carretera Tijuana-Ensenada, 22860
Ensenada, Baja California, Mexico
A R T I C L E I N F O
A B S T R A C T
Keywords:
Phosphonates-based agents are well-known bone-seeking radiopharmaceuticals with application in detection
and therapy. With higher sensitivity and resolution offered by Positron Emission Tomography (PET), tracers
Bone imaging
PET/CT
68
based on this technique are gaining huge attention. Ga-based generator and radiotracers render independence
Gallium-68
DOTA
68
from the on-site cyclotron. We report the development of Ga-labeled DOTA-based bismacrocyclic phosphonate
68
derivative, for bone PET imaging. The synthesis and characterization of Ga- DO3P-AME-DO3P was carried out
Phosphonates
Skeletal metastases
in > 95% purity. The radiotracer displayed high stability and low binding affinity (< 3%) to blood serum. High
in vitro binding affinity were observed for synthetic hydroxyapatite, SAOS-2, osteoclast and osteoblast cells. In
vivo pharmacokinetics revealed fast washout with biphasic release pattern. The deposition of radiotracer in
osseous tissues was high (Bone/Muscle ratio:18), as studied from the biodistribution studies. In vivo PET/CT and
6
8
biodistribution analyses revealed the ability of Ga-DO3P-AME-DO3P to target and accumulate in bone, thus
displaying its potential as a PET bone imaging agent.
1
. Introduction
Skeletal metastases are the most frequent malignancies caused by
analogs of naturally occurring pyrophosphates with a high affinity to-
wards calcified tissue and are the choice of biovectors for diagnosis and
treatment of bone disorders [4,5,10]. However, the phosphonate groups
9
9m
the invasion of primary cancers to bone [1,2]. Patients with advanced
prostate or breast cancers are most likely to develop bone metastasis,
constituting around 75% of the reported cases [3]. Once established, it
strongly reduces the survival rates and causes severe complications like
bone pain, impaired mobility, spinal cord compression, pathological
fractures, and hypercalcemia [1,2,4]. Bone metastases disturb the bone
homeostasis mediated by osteoblasts and osteoclasts, leading to an in-
crease and decrease in bone lesions, respectively [4]. Early assessment
of metastatic turnover is clinically important for disease prognosis and
determining the optimum treatment strategy to inhibit the pathological
fractures from disabling the patients [5]. Despite the significant ad-
vancement in the understanding of molecular biology of skeletal me-
tastases, the early diagnosis remains challenging, and the condition
continues to be a devastating complexity of advanced cancer.
of
Tc-MDP bind to the calcium of crystalline hydroxyapatite (HA)
99m
present in the bone matrix as well as the radiometal
reduce the inherent adsorption of MDP.
Tc, which may
1
8
18
Positron emission tomography (PET) using F-sodium fluoride ( F-
NaF) offers superior diagnostic resolution and enhanced sensitivity in
bone imaging [11,12]. The higher sensitivity and resolution provided
by PET have led to an increased interest in PET radiopharmaceuticals,
which can be developed through simple strategies. The emergence of
6
8
Ga-generators allowed to integrate the benefits of the high sensitivity
of PET with the ligand-based tracers to exhibit a remarkable improve-
6
8
ment in imaging accuracy. The on-site Ga-generators have provided
independence from the cyclotrons, and multiple radiosynthesis proce-
1
8
68
dures of F-based tracers, have prompted extensive utilization of Ga-
based bifunctional chelate conjugated with biovectors for routine
imaging [13,14]. Bone PET imaging is being performed by a variety of
acyclic and macrocyclic chelators modified by phosphonate ester moi-
9
9m
Radionuclide based bone scintigraphy using
Tc-phosphonates
9
9m
like methylenediphosphonate ( Tc-MDP) are the mainstay for de-
tection of the status of bone metabolic activity after tumor invasion
6
8
eties, which introduce bone-seeking properties [4,5]. The Ga-com-
plexes with acyclic chelator ethylenediamine tetramethylene
[
6–9]. Phosphonates-based agents are the biologically stable synthetic
⁎
Corresponding authors.
E-mail addresses: akmishra@inmas.drdo.in (A.K. Mishra), anupama@inmas.drdo.in (A. Datta).
https://doi.org/10.1016/j.bioorg.2020.104185
Received 16 March 2020; Received in revised form 21 June 2020; Accepted 28 July 2020
Available online 26 August 2020
0045-2068/ © 2020 Elsevier Inc. All rights reserved.

K. Chauhan, et al.
Bioorganic Chemistry 104 (2020) 104185
phosphonate (EDTMP) and diethylenetriamine pentamethylene phos-
phonate (DTPMP) display high bone uptake [15]. Yet, their clinical
prospects remained speculative due to the low in vivo stability of the
tracers with poor therapeutic utility.
assays using human blood samples were approved by the institutional
ethical committee. The samples from healthy volunteers were collected
after providing them information and obtaining written consent. All the
participants were above 18 years of age.
The macrocyclic chelators, α-aminomethylphosphonates, viz, DOTP
are extensively used for the diagnosis of bone cancer and is established
Synthesis of 1,4,7,10-tetraazazyclododecane tetramethylenepho-
sphonic acid (DOTP): The monomeric analogue, DOTP (1,4,7,10-tetra-
azazyclododecane tetramethylenephosphonic acid) was synthesized by
converting the carboxylic functions of DOTA into phosphonic functions
using phosphorous acid and phosphorus trichloride with a 90% yield and
high purity [16].
1
66
for therapeutic purposes with radiometals (β-emitters) like
Ho,
1
53
186
Sm and
Re [16–22]. The kinetic inertness of DOTA-based chela-
tors with different metal ions in the + 3 oxidation state is well docu-
mented [19,14,23]. Thus, the substitution of β+ by β-/α-particles is
also possible, which provides theranostic ability at the same platform.
Further, 10′-bis(acetamido)-ethane-bis[1,4,7-tri(methylene phosphonic
2.3.1. Synthesis of 10′-bis(acetamido)-ethane-bis[1,4,7-tri(methylene
phosphonic acid)-1,4,7,10-tetraazacyclododecane] (DO3P-AME-DO3P)
The synthesis of bismacrocyclic DO3P-AME-DO3P was performed
by coupling of two DO3P moieties with the help of ethylene diamine as
linker [23]. (Fig. S1). The main synthetic steps are as follows:
Tert-butyl groups of ligand, 10ʹ-bis(acetamido)ethane-bis[1,4,7-tri(car-
bobutoxymethane)]-1,4,7,10-tetraazacyclododecane (1 mmol) were depro-
tected with neat trifluoroacetic acid (3 mL) at 0 °C to give DO3A-AME-
DO3A, (10ʹ-bis(acetamido)-ethane-bis[1,4,7-tri-(carboxymethane)-1,4,7,10-
tetraazacyclododecane]) in 84.9% yield after purification. DO3A-AME-
DO3A (9.49 mmol) was further converted into its phosphonate derivative,
DO3P-AME-DO3P by using phosphorous acid (40 mmol) and phosphorus
trichloride (0.44 mmol). The reaction was carried out in toluene (10 mL) at
90 °C for 6 h. Toluene was evaporated under vacuum and crude product
obtained was dissolved in water and precipitated in methanol. The com-
pound was purified by HPLC in 90% yield.
acid)-1,4,7,10-tetraazacyclododecane], DO3P-AME-DO3P
a
multi-
dentate bismacrocyclic phosphonate ligand, augment the bone binding
potential with its higher number of phosphonate groups. SPECT
9
9m
radiocomplex,
Tc-DO3P-AME-DO3P, displayed better binding to-
wards synthetic hydroxyapatite and in vivo bone uptake compared to
monomacrocyclic phosphonate system, DOTP. Additionally, the ligand
3
+
3+
3+
has high stability with different metal ions (like Sm , Ho , Lu ) of
therapeutic value [23].
6
8
With the current demand for new
Ga bone-seeking radio-
pharmaceuticals and the encouraging results of DO3P-AME-DO3P in
SPECT, we investigated the efficacy of the ligand as a cost-effective PET
6
8
imaging radiotracer for bone metastases after radiolabeling with Ga.
The present study includes the preliminary assessment of DO3P-AME-
DO3P for its potential role as a PET bone imaging agent. The different
6
8
parameters for Ga-labeling of DO3P-AME-DO3P are optimized to
obtain a high radiochemical yield, and the consequent radiocomplex is
evaluated for its stability under physiological conditions. The phar-
2.3.2. Cold labeling of DO3P-AME-DO3P with 6
9/71
Gallium
6
8
macokinetics and bone-seeking potential of Ga-DO3P-AME-DO3P was
evaluated in vitro and in vivo and compared with the corresponding
DO3P-AME-DO3P was complexed with stable Ga isotope, according
to our previous report [13]. Briefly, DO3P-AME-DO3P and Ga
6
8
Ga-DOTP complex.
3
(NO )
3
·xH O were dissolved in sodium acetate buffer (pH 4.5) in 1 : 1
2
stoichiometric ratio and the reaction mixture was stirred at 70 °C for
30 min. The reaction progress was monitored by MS/ESI. The complex
formed was lyophilized overnight to give a white powder (94% yield).
The chemical purity was determined by HPLC [Agilent 1200 LC, C-18
2
. Materials and methods
2.1. Chemicals
RP Agilent column (5µ, 1 mm X 125 cm)].
+
1
,4,7,10-Tetraazacyclododecane was purchased from Sterm
ESI-MS (m/z) calculated: [M] 1184.12 ; found 1184. 7045
.
HPLC:
Chemicals Limited, USA. All other chemicals were commercially ob-
tained as a reagent grade from Sigma Aldrich or Merck, Germany, and
used without purification. The thin-layer chromatography (TLC) ana-
lyses were performed to monitor the reactions using silica gel 60 F254
plate obtained from Merck. Silica gel MN60 (60–200 μm, Aldrich) was
used for purification of intermediate products of multi step synthesis of
(λ = 214 nm; 10% 10 mM ammonium acetate and 90% acetonitrile,
flow rate 1.5 mL/min; t = 8.408 min.
R
2.4. Radiolabeling studies with 68Ga
The fresh 68Ga activity (t1/2 = 68 min, β+ = 89% and EC = 11%)
6
8
68
68
final ligand DO3P-AME-DO3P [23]. Ga (t1/2 = 68 min) was eluted
was eluted from the Ga/ Ge-generator (760 MBq) with 0.1 N HCl
(6 mL) and was trapped on a cation exchange Strata X-C cartridge
column. The column was eluted with 80% acetone-0.15 N HCl (1 mL),
6
8
68
from in house Eckert and Zieglar Ge/ Ga generator system (IGG100,
Berlin).
6
8
and complete desorption of the purified GaCl was achieved with 98%
3
2.2. Instrumentation
acetone-0.05 N HCl mixture (500 μL). The ligand (30–50 μM) was
dissolved in sodium acetate solution (0.2 M) and passed through a
0.22 μm syringe filter. The reaction mixture containing ligand and
The synthesized compounds were characterized by nuclear mag-
netic resonance (NMR) and mass spectrometry using the Bruker Avance
purified GaCl (400 μL) was incubated for different time intervals
3
1
13
II spectrometer ( H NMR 400 MHz and C NMR 100 MHz) and Agilent
(0–20 min) at varying temperatures and pH. Thereafter, the mixture
was diluted with water (1 mL) and passed over a weak anion exchanger
cartridge. The loaded cartridge was first washed with water (1 mL), and
the final tracer was eluted with phosphate buffer saline (PBS 2 mL). The
radiochemical efficiency was deduced through ITLC run in a 1:1 ratio of
6
310 ion trap mass spectrometer respectively. The radiochemical yield
and purity were checked by ITLC-SG (Paul Gel-man, USA) and scanned
using omniscan EZ-TLC scanner. Radioactivity studies were carried out
using a γ-scintillation counter GRS230, ECIL (India). PET/CT scan was
executed on a Discovery STE 16 (GE) system.
6
8
1 M ammonium acetate: methanol. The percentage of free Ga and
6
8
Ga-complex was calculated. The TLC strip of the final complex was
2.3. Animal models
scanned on omniscan EZ-TLC scanner. The identity of the complex was
checked through radio HPLC and the molar activity was 9.3–9.8 MBq/
nmol after purification.
The bone imaging and blood clearance studies were performed in
6
8
New Zealand rabbits (weighing 2–3 kg), and the biodistribution ana-
lysis was carried out using BALB/c mice (22–28 g). The animal proto-
cols were approved by the Institutional Animal Ethics Committee (Reg.
no. 8/GO/a/99/CPCSEA). The haemolysis studies and serum stability
The similar procedure was followed to synthesise Ga-DOTP and
68
the carboxylic acid complex Ga-DO3A-AME-DO3A. The estimated
6
8
68
molar activity of Ga-DO3A-AME-DO3A and Ga-DOTP after pur-
ification were 9.8–10.2 and 7.8–8.2 MBq/nmol, respectively.
2

K. Chauhan, et al.
Bioorganic Chemistry 104 (2020) 104185
2.5. In vitro stability evaluation in the presence of endogenous metal ions
mmolL-1 (2.88–33.6 kBq). The prepared solutions were gently shaken
for 20 min. The suspension was then filtered through Millipore filter
(0.22 μm), and 1.0 mL of the filtrate was counted.
The percentage bound activity for the binding assay was calculated
as equation (1):
6
8
Ga-DO3P-AME-DO3P was prepared under optimum conditions,
and the complexation yield was calculated. The radiocomplex was
challenged with the metallic species (Fe, Cu, Zn, and Ca) in 1:103 M
ratio of radiocomplex/metal ion. The percentage of intact tracer was
A(%) = [(cin cfi)/cin] × 100%
(1)
6
8
calculated as a function of time after incubation of Ga-DO3P-AME-
DO3P in the presence of excess metal ion at 25 °C with the help of ITLC.
where cin, c denote the initial and final radioactivities, respectively,
fi
and A% is the adsorption percentage of complex.
2.6. Human serum stability evaluation
2.9. Cell experiments
The evaluation of 68Ga-DO3P-AME-DO3P stability in human serum
and the serum protein binding assay was performed [24]. The blood
samples obtained from the healthy volunteers were allowed to clot for
Cytotoxicity studies: Cytotoxicity of the synthesized compound was per-
formed on human cells, HEK-293 (Human Embryonic Kidney 293) procured
from National Centre for Cell Science, Pune, India) using XTT. The selected
compounds were tested in a concentration range of 5 nM-0.5 mM for 12,
24, and 48 h. The HEK-293 cells were cultured in high glucose DMEM
supplemented with 10% fetal bovine serum and 1% (v/v) penicillin strep-
4
5 min at 37 °C in a humidified incubator at 95% air / 5% CO . The
2
serum was separated by centrifugation at 400 g by filtration through a
6
8
0
.22 µm syringe filter. The Ga-DO3P-AME-DO3P (100 µL) was mixed
with human serum (900 µL) and incubated at 37 °C for 240 min. The
percentage of free gallium was analyzed by running ITLC-SG in the
mixture of 1 M ammonium acetate/methanol (1:1) at different time
intervals till 240 min.
4
tomycin. The cells were seeded at ~1 × 10 /well with a final volume of
250 µL in 96-well plates with or without the ligand. The plates were in-
cubated at 37 °C in a 5% CO incubator for different time periods. At the
2
end of incubation, 100 µL of XTT (2, 3-bis (2-methoxy4-nitro-5-sulfo-
phenyl)-5-[(phenylamino) carbonyl]-2Htetrazolium hydroxide) solution
mix was added to the solutions in well, and plates were incubated at 37 °C
for another 2 h The optical density is measured at 450 nm, and mi-
tochondrial activity was calculated as % metabolic viability.
2.7. Haemolysis assay
The ligand was investigated for its haemolytic activity in ac-
cordance with established protocols [25]. Fresh blood collected from
healthy volunteers in heparinized tubes was centrifuged at 4 °C for
Binding studies with SAOS-2 cells: The binding affinity of the radi-
oligand was performed with SAOS-2 that were procured from the
National Centre for Cell Science, Pune, India. The culture of the SAOS-2
cells was performed as described [23]. For the binding studies, the
washed cell line culture was incubated for 40 min with an increasing
1
0 min at 3000 rpm. The resultant RBC pellet after removal of plasma
was washed thrice with PBS (pH 7.4) by the process for resuspension
and centrifugation (3000 rpm, 10 min, 4 °C). Erythrocytes were finally
resuspended in PBS and aliquots of equal volume were incubated with
the ligand samples of varying concentrations in the ratio of 8:2. The
samples were incubated at 37 °C and time-dependent haemolysis of the
samples was studied at time points of 15 min, 1 h, 2 h, 4 h, and 24 h.
After the incubation, suspensions were centrifuged as mentioned pre-
viously, and supernatants were studied for the extent of haemolysis.
Synergy 2 Multi-Mode Reader (BioTek Instruments, USA) was used to
study the released haemoglobin in the supernatant by measuring op-
tical density at 540 nm. Triton X-100 (2%v/v) served as positive control
and corresponded to 100% haemolysis, whereas PBS was regarded as
the negative control. The experiment was conducted in triplicates and
the percentage of erythrocyte haemolysis was determined.
6
8
concentration of Ga- DO3P-AME-DO3P in the presence and absence of
100-fold DO3P-AME-DO3P to estimate the total binding and non-
specific binding. Afterward, cells were washed with ice-cold binding
buffer three times for 3 min. The cells were lysed with lysis buffer, and
the cell-associated radioactivity counts were determined. Scatchard
plot analysis was carried out.
Cell studies on osteoblast and osteoclast cells: Osteoblast cells were
isolated from calvarias of newborn mice, while the osteoclast cells were
isolated from 5 to 10 weeks old mice. The procedure followed for the
isolation and culture of the cells is followed according to the reference
[23].
In vitro cell uptake: In a 24-well plate containing the cell culture, the
6
8
2.8. In vitro binding studies with hydroxyapatite
medium was removed, and Ga-DO3P-AME-DO3P (37 kBq/mL; total
volume 0.5 mL using fresh R-MEM plus 10% FBS) was added to each
well. The cells were incubated, and cells were washed with serum-free
medium (1 mL) at different time points. The cells were lysed with Tris-
buffered saline (150 μL) containing detergent. The lysates were then
Estimation of the time required to attain thermodynamic equilibrium: In
a vial, HA (50 mg) was suspended in a TRIS buffer solution (0.10
-
1
68
mmolL , pH = 7.5). To this Ga-DO3P-AME-DO3P complex was
-
1
68
added (final concentration of tracer was 9.45 kBq, 1.00 mmolL and
the total volume of solution was 4.0 mL), and the resulting solution was
gently shaken. After incubation (5 min, 15 min, 30 min, 45 min,
transferred to microcentrifuge tubes, and uptake of Ga-DO3P-AME-
DO3P was measured by counting the cell lysates in γ-counter. The
protein concentration in each lysate was determined.
6
0 min, 75 min, 2 h & 4 h; one vial for each point), the suspension was
filtered through Millipore filter (0.22 μm), washed twice with TRIS
buffer (1 mL), and counts in 2 mL of the filtrate were checked. The
experiment was carried out in triplicate. The adsorption behavior of
2.10. Blood kinetics
The blood pharmacokinetics of the developed radiocomplex was
evaluated according to our previous report [26]. The radiotracer
(200 µL, 37 MBq) was intravenously injected through the dorsal ear
vein of a normal rabbit. The blood samples (200 µL) were withdrawn at
different time intervals. The activity persistence in terms of percentage
administered dose in the collected samples was counted using a well
counter and plotted as a time-activity curve.
6
8
Ga-DOTP on hydroxyapatite was also evaluated under the same
conditions.
Competitive sorption of ligand DO3P-AME-DO3P and 68Ga-DO3P-AME-
DO3P complex on HA: In a 10 mL vial, HA (50 mg) was suspended in a
-
1
68
TRIS buffer solution (0.010 mmolL , pH = 7.5). A solution of Ga-
DO3P-AME-DO3P and ligand DO3P-AME-DO3P were prepared. Three
sets of experiments were performed with varied concentrations of
6
8
radiocomplex and free ligand solution. Experiment A: Ga-DO3P-AME-
2.11. Imaging studies
-
1
-1
DO3P 0.7 mmolL (6.72 kBq), DO3P-AME-DO3P 0–1.5 mmolL ; ex-
6
8
-1
periment B: Ga-DO3P-AME-DO3P 1.0 mmolL (9.6 kBq), DO3P-AME-
The in vivo PET/CT scan was performed on the healthy rabbits
-
1
68
68
DO3P 0–1.1 mmolL ; experiment C: Ga-DO3P-AME-DO3P 0.3–3.5
(n = 2). The animal was injected intravenously with ~ 34 MBq of Ga-
3

K. Chauhan, et al.
Bioorganic Chemistry 104 (2020) 104185
DO3P-AME-DO3P through the dorsal ear vein. The imaging was then
performed at different time points using a PET camera. The 16-slice
PET/CT system with a spatial resolution of 5.0 mm in all directions and
an axial field of view (AFOV) of 15 cm was used. It operates in 2D as
well as 3D mode. For the present study, the data were acquired in 3D
mode. PET protocol involving dynamic acquisition of temporal images
was used. Dynamic PET images were acquired in three different regions
of the body for different time interval at the rate of five min per frame
so as to study variation in activity with respect to time in six organs
namely brain, liver, spleen, kidneys, bone and stomach. The images
were reconstructed using a fully 3D ordered subset expectation max-
imization (3D-OSEM) algorithm with all corrections (scatter, random,
dead time, attenuation and normalization) incorporated into the
iterative reconstruction scheme. The quantification of uptake in bone
and other organs was done by ROI analysis.
chelate may dissociate in the presence of the endogenous metal ions
present in a considerable concentration in the blood plasma. The dis-
6
8
3+
sociated Ga
may accumulate in non-targeted organs or give false
68
positive information. Therefore, in vitro competitive assay of Ga-
3
DO3P-AME-DO3P was carried out against 10 times molar excess of
2
+
2+
2+
3+
metal ions of physiological relevance (Ca , Zn , Cu , and Fe ).
The purity of the complex was assessed as a function of time after
incubation of the complex in the presence of metal ions at room tem-
6
8
perature up to 270 min (4 × t1/2 of Ga). The radiocomplex was stable
in the presence of metal ions until 150 min; however, a slow de-
gradation was observed at the later time points (Fig. S4). The overall
stability of the complex at 120 min was 97, 95.4, 97, and 94.6% in the
3
+
2+
2+
2+
,
presence of Fe , Zn , Cu , and Ca
respectively.
6
8
The in vitro stability studies of Ga-DO3P-AME-DO3P were per-
formed in freshly extracted human serum under physiological condi-
tions. The strong interaction of radiotracers with serum proteins is
undesirable, and their enzymatic cleavage in human plasma can
2.12. Biodistribution
6
8
markedly lower their potential. After the incubation of Ga-DO3P-
AME-DO3P in the blood serum up to 270 min at 37 ˚C, the percentage of
intact tracer was > 94%. Also, the binding behavior of the tracer with
proteins (albumin and transferrin) present in serum exhibited
2.8 ± 0.3, 2.9 ± 0.6 and 3.2 ± 0.1% binding at 10, 60 and 120 min,
respectively. The study suggests the low binding of the tracer with
serum proteins and the suitability of tracer for in vivo application.
The normal BALB/c mice (n = 5) were administered with 68Ga-
DO3P-AME-DO3P (100 µL, 3.5 MBq) through intravenous injection to
the tail vein. At different time intervals, the mice were sacrificed via
cardiac puncture and dissected. The desired organs were removed,
rinsed with cold saline, weighed, and radioactivity was counted on a
well counter. The data are expressed as percentage injected dose per
gram of organ (% ID/g).
3.3. Haemolysis studies
3
. Results and discussion
For the intravenous mode of administration, the blood affinity of the
3.1. Synthesis and radiolabeling
ligand is an important aspect. Haemolysis occurs due to the affinity of
the ligand towards the proteins present in the erythrocyte membrane. A
higher level of interaction leads to the rupture of RBCs, invariably
causing higher levels of toxicity. Consequently, haemolysis assay for
DO3P-AME-DO3P was performed at varying concentrations to quantify
membrane damage to RBCs. It was observed that DO3P-AME-DO3P did
not induce any apparent haemolysis at either of the concentrations up
to 4 h (% haemolysis < 10%). Haemolysis by the ligand at any time
point was comparable to the haemolysis value of the negative control
(Fig. S5). The above results establish the nontoxic nature of ligand for
erythrocytes, thereby rendering it suitable for in vivo purpose.
The bismacrocyclic, DO3P-AME-DO3P, was synthesized by the
coupling of two DO3P units via a linker based on ethylenediamine,
according to our previous report (Fig. S1) [23]. The complexation of
6
9/71
DO3P-AME-DO3P with the stable gallium isotope (
Ga) was con-
firmed by mass spectrometry. The molecular weight of 1184 g/mol
proved the complexation of gallium to DO3P-AME-DO3P. The frag-
nat
mentation pattern of the Ga-conjugate ligand was similar to the ex-
6
9
pected isotopic pattern of stable gallium isotope, which contains Ga
7
1
(
60.11%) and Ga (39.89%) (Fig. 1) [27] the HPLC profile.
3+ 68 68
The Ga
achieved in less than five minutes. The radiolabeling parameters
temperature, pH, and heating period) of DO3P-AME-DO3P with
elution from Ga/ Ge-generator and processing was
3.4. Adsorption studies on hydroxyapatite
(
6
8
3+
Ga
were optimized (Fig. S2), and the radiolabeling efficiency was
The bone targeting efficiency was first tested by in vitro binding
analysis on synthetic hydroxyapatite, which is a major constituent of
bone mineral. Hydroxyapatite serves as a valuable model for obtaining
information about the ligand interaction with bone and understanding
the ligands uptake kinetics in an independent environment. The ad-
sorption kinetics on preconditioned hydroxyapatite was studied
through the batch method, and the amount of radioligand present in the
suspension was measured after specific time intervals. The binding
monitored by ITLC [14]. The maximum yield of ~ 93% was achieved
6
8
after 10 min heating at 60-65˚C. The formation kinetics of Ga-DO3P-
AME-DO3P at other temperatures was lower, and the maximum radi-
3
+
olabeling efficiency was in the range of 50–84%. As Ga
forms Ga
–
37
(
OH) (Ksp = 5 × 10 ) at higher pH > 7,[29], the labeling reaction
3
was carried out at pH 4.0–5.0 to ensure the suppression of Ga(OH)
3
formation. The radiochemical yield reached a plateau within 10 min.
The final radiocomplex was obtained with > 95% purity. The for-
mation of radiocomplex was demonstrated by ITLC studies where
6
8
potential of Ga-DO3P-AME-DO3P was compared with the acid pre-
6
8
cursor, Ga-DO3A-AME-DO3A. The non phosphonate ligand displayed
low binding (8% in 10 min; 15% in 1 h) on hydroxyapatite. Contrarily,
radioactivity was observed at the R of 0.8–0.9 which corresponded to
f
6
8
68
Ga-DO3P-AME-DO3P, while no peak at R
f
0.0–0.1 suggested absence
Ga-DO3P-AME-DO3P showed fast uptake with ~46% bound activity
3
+
of free Ga . The molar activity was estimated as 9.3–9.8 MBq/nmol
after purification. The HPLC analysis further confirmed the chemical
identity and purity of the radiocomplex. Under similar conditions, the
at 10 min incubation and reached to maxima (~91%) within 1 h of
incubation (Fig. 2). Insignificant increase in the sorption on hydro-
xyapatite with time was observed at the later time points. The observed
6
9/71
68
Ga-DO3P-AME-DO3P elutes out at 8.408 min while the radio-HPLC
binding was higher than the corresponding monocyclic analog, Ga-
analysis showed a single peak at retention time 8.378 min thus, con-
firming the formation of similar chemical species i.e, the radio complex
DOTP, thus, may be directly related to the increase in the number of
6
8
phosphonate groups. Furthermore, the percentage uptake of Ga-
6
8
99m
Ga-DO3P-AME-DO3P.
DO3P-AME-DO3P was similar to the
Tc-labeled analogue suggesting
that the uptake was primarily due to the interaction of phosphonic acid
with hydroxyapatite irrespective of the choice of radiometal ion. The
3.2. In vitro stability and serum protein binding assay
6
8
sorption phenomenon of Ga-DO3P-AME-DO3P was found to be re-
Metal ions like Ca2 , Zn , Cu , and Fe
+
2+
2+
3+
are usually present in
versible when the hydroxyapatite adsorbed with Ga-DO3P-AME-
68
human serum. After the tracer administration in the body, the metal
DO3P was treated with free DO3P-AME-DO3P.
4

K. Chauhan, et al.
Bioorganic Chemistry 104 (2020) 104185
Fig. 1. Mass spectra of 10′-bis(acetamido)-ethane-bis[1,4,7-tri(methylene phosphonic acid)-1,4,7,10-tetraazacyclododecane] bis Gallium (III) 69/71Ga-DO3P-AME-
DO3P. Calculated mass [M]+ 1184 found m/z = 1184 [M]
+.
Fig. 2. In vitro binding assay of 68Ga-DO3P-AME-DO3P and Ga-DOTP with
68
synthetic hydroxyapatite at 1 h. The results are expressed as mean
±
SD
(
n = 3).
Fig. 3. Competitive sorption experiment of the ligand DO3P-AME-DO3P and
3
.5. The competitive co-sorption experiment
68
68
Ga-DO3P-AME-DO3P complex: (+) initial concentration: Ga-DO3P-AME-
-
1
-1
DO3P 0.7 mmolL , DO3P-AME-DO3P 0–1.5 mmolL , (o) initial concentration:
The competitive co-sorption binding assay between 6 Ga-DO3P-
8
68
-1
-1
Ga-DO3P-AME-DO3P 1.0 mmolL , DO3P-AME-DO3P 0–1.1 mmolL , (Δ)
initial concentration: 68Ga-DO3P-AME-DO3P 0.3–3.5 mmolL
-1
.
AME-DO3P and DO3P-AME-DO3P were performed at different con-
-
1
centrations of labeled ligand and free ligand. When 1.5 mmolL of
6
8
Ga-DO3P-AME-DO3P was added for sorption on HA, activity left on
the supernatant after 30 min was found to be identical to the super-
3.7. Bone cellular uptake studies
-
1
68
natant obtained from the treatment of 0.7 mmolL of Ga-DO3P-AME-
The phosphonate based bone imaging agents allow visualization of
-
1
DO3P and 0.8 mmolL of DO3P-AME-DO3P (Fig. 3). The competitive
co-sorption experiments reveal almost similar activity in the solutions
for all ratios of free ligand and complex used for the study. The study
indicates no significant changes in the binding property, capacity, and
phenomenon of the ligand upon complexation with 68Ga.
both normal and abnormal bone metabolism processes which are
evaluated in terms of bone turnover. Whenever, any abnormal meta-
bolism leads to metastatic lesion of osteoblastic and osteolytic types,
the agent is able to diagnose the event. The osteoblastic lesions are
involved in bone formation and are less threatening to bone mor-
phology and its health. Contrastingly, the osteolytic lesions character-
ized by normal bone destruction cause serious clinical problems and
greatly affect the bone health. These osteolytic lesions are over-
3
.6. Receptor binding studies
9
9m
expressed with osteoclasts cells. The routinely used
Tc-MDP in
The SAOS-2 cells display characteristics similar to osteoblast. The
clinics detects the osteoblastic activity efficiently; however, osteoclastic
activity is poorly diagnosed. Therefore, currently the focus is towards
the imaging agents that can visualise the osteolytic lesion. The accu-
6
8
receptor binding assay indicates the binding affinity of the Ga-DO3P-
AME-DO3P complex with SAOS-2 cells. The Scatchard analysis sug-
gested significant binding of the labeled DO3P-AME-DO3P conjugate
68
mulation of Ga labeled complex was tested in isolated osteoblasts and
with K of 1.84 nM (Fig. S6).
d
5

K. Chauhan, et al.
Bioorganic Chemistry 104 (2020) 104185
Fig. 4. Uptake studies of 68Ga-DO3P-AME-DO3P in osteoclast (OC) and osteo-
blast (OB) cells. The results are expressed as mean ± SD (n = 3).
6
8
Fig. 6. Bone imaging of a healthy rabbit using Ga-DO3P-AME-DO3P at
5 min p.i. A) Coronal view; and B) Transaxial view of PET, CT and PET/CT.
osteoclasts cells. The study with osteoblasts and osteoclasts bone cells
showed accumulation up to 100.80 ± 2.29% dose/mg protein and
4
1
60.41 ± 2.94% dose/mg protein after 50 min of incubation, re-
observed. The trend for blood clearance was lower than the previously
spectively. The cellular uptake was dependent on the time of incubation
9
9m
reported
Tc-DO3P-AME-DO3P [t1/2(F) ~ 15 min and t1/2(S) ~ 4 h];
Tc-MDP [t1/2(F) ~ 55 min and t1/
(
Fig. 4) and was highest in the pH range 6.2–7.3. Many bone diseases
9
9m
however, it was faster than
like cancer-associated or osteoporosis show enhanced bone resorption
by osteoclasts. Thus, the ligand can be applied in different bone-related
disorders.
2
(S) ~ 7 h 55 min] [23]. A rapid washout of the radiotracer may be
beneficial for the early imaging after administration and also lowers the
radiation burden to the subject [24,28].
3.8. Cytotoxicity studies
3.10. In vivo PET/CT and biodistribution studies
For an ideal imaging agent, good safety profile, i.e., minimal toxi-
The in vivo PET/CT imaging was performed on healthy rabbits
city, is requisite; therefore, in vitro cytotoxicity of the ligand was as-
sessed by incubating normal healthy HEK-293 cells with different
concentrations (nM-mM) for different time intervals (12 h, 24 h, and
(
n = 2) using an integrated PET/CT scanner at different intervals of
68
time. Ga-DO3P-AME-DO3P exhibited high accumulation on animal
skeletal within 1 h and reached the maxima in 2 h (Fig. 6). From the
coronal and the sagittal view, the tracer concentration was clearly
visible in bone joints, shoulder and spinal cord, which represent the
metabolic active regions of bone. After PET/CT coregistration, a more
explicit representation of the uptake in skeletal was seen. The quanti-
fication of bone uptake was realized by regions of interest (ROI) ana-
lysis and was ~ 30 times higher than the uptake in muscles.
4
8 h). The cytotoxicity effects of DO3P-AME-DO3P were evaluated with
XTT colorimetric assay on HEK cells. The graph between the surviving
fraction and concentration of the ligand at different time points is
shown in Fig. 5. The ligand displayed low toxicity (50 nM, 96.4%
survival at 24 h, 500 nM, 92% at 24 h), suggesting the suitability of
radioligand as a safe diagnostic agent.
9
9m
Previous studies have investigated the
Tc complex of DOTP for
3
.9. Blood kinetics studies
SPECT imaging or pretherapy quantitative diagnostic scanning, which
6
8
displayed encouraging results and was later studied with Ga-labeling
The blood pharmacokinetics of 68Ga-DO3P-AME-DO3P was per-
68
[
16,29]. The Ga-complex had high hydroxyapatite binding with
formed in healthy rabbits. The tracer exhibited 56% residual activity in
blood at 30 min and 29% at 2 h. Fast clearance of activity from the
blood was observed during the initial time point of 30 min, and the
washout trend was then slow (Fig. S7). A biphasic pattern with t1/
moderate bone uptake in rats; however, it was obtained in low labeling
68
yields [29]. DOTA-based conjugate, Ga-BPAMD exhibited rapid lo-
calization in the bone [30]. However, detailed studies with regard to
68
biological distribution in the different organs of these Ga complex
2
(F) = 21 min ± 0.025 min, t1/2(S) = 5 h 17 min ± 11 min was
were not reported, which is useful for correct predictions of the prop-
erties.
The distribution of 68Ga-DO3P-AME-DO3P in different organs was
carried out by performing the biodistribution studies in mice, and
percent injected activity (% IA) of each organ was determined based on
the activity measured per gram of organ or tissue (Table 1). Similar to
6
8
imaging study, the uptake of Ga-DO3P-AME-DO3P in bones was fast
9
9m
and reached a maximum in 2 h. When compared with
Tc complex,
6
8
Ga-DO3P-AME-DO3P took a longer time to accumulate in the bone,
which could be attributed to slower pharmacokinetics of the Ga com-
plex. Consistent with the hydroxyapatite binding studies, the in vivo
6
8
68
bone uptake of Ga-DO3P-AME-DO3P was higher than Ga-DOTP,
suggesting an improvement in binding affinity with the increase in
6
8
phosphonic acid groups. The high bone to muscle ratio of Ga-DO3P-
6
8
AME-DO3P (B/M:18) over Ga-DOTP (B/M:7) further highlights the
impact of high denticity in the form of phosphonate group per ligand.
9
9m
The uptake in the liver was low. In comparison to the
Tc-analogue,
Fig. 5. In vitro cytotoxicity analysis of DO3P-AME-DO3P with healthy HEK-293
the bone uptake in terms of %ID/g is same however, bone to muscle
cells. The results are expressed as mean ± SD (n = 3).
99m
ratio improved from 9 to 18. While
Tc-MDP reached to maximum
6

K. Chauhan, et al.
Bioorganic Chemistry 104 (2020) 104185
2
+
2+
2+
3+
Table 1
endogenous metal ions, Ca , Zn , Cu , and Fe . The binding
6
8
68
68
Biodistribution analyses of Ga-DOTP and Ga-DO3P-AME-DO3P in normal
mice. The data is the mean percentage (n = 3) of the injected dose of radio-
tracer per gram of organ, ± the standard deviation of the mean.
analysis of Ga-DO3P-AME-DO3P with synthetic hydroxyapatite de-
6
8
monstrated higher binding compared to the monocyclic Ga-DOTP.
The binding affinity (K ) of the ligand towards SAOS-2 cells was ob-
d
6
8
Ga-DOTP
served in the nanomolar range. While, the cytotoxicity analysis in
normal HEK cell lines revealed the relatively safe nature of the ligand.
1
h
2 h
4 h
68
The uptake of Ga-DO3P-AME-DO3P in bone reached a maximum at
2
h with a bone to muscle ratio of 18. The potential of the ligand as an
Blood
3.21 ± 0.36
1.87 ± 0.11
2.02 ± 0.46
2.6 ± 0.18
1.48 ± 0.06
8.22 ± 0.55
1.62 ± 0.11
2.75 ± 0.23
1.3 ± 0.03
4.7 ± 0.28
1.46
2.6 ± 0.35
1.22 ± 0.20
1.48 ± 0.31
2.35 ± 0.32
1.33 ± 0.15
5.79 ± 0.61
1.27 ± 0.17
1.78 ± 0.10
0.72 ± 0.02
5.23 ± 0.69
2.01
1.02 ± 0.07
0.67 ± 0.05
0.94 ± 0.26
2.08 ± 0.24
1.15 ± 0.12
3.64 ± 0.35
1.14 ± 0.08
1.36 ± 0.13
0.68 ± 0.10
4.8 ± 0.36
4.7
Heart
effective and feasible PET imaging agent with therapeutic promise was
further substantiated by in vivo PET imaging and biodistribution stu-
dies, which revealed high bone accumulation. The increased efficiency
of dimeric system and preferential uptake in osteoclasts in comparison
to osteoblasts coupled with the already reported high stability of DO3P-
AME-DO3P with the therapeutic radiometals offers a “2 in 1” ther-
anostic approach for bone metastases, which is of significant commer-
cial interest as PET bone-seeking agent.
Lungs
Liver
Spleen
Kidney
Stomach
Intestine
Muscle
Bone
Bone/Blood
Bone/Muscle
3.61
7.26
7.05
6
8
Declaration of Competing Interest
Ga-DO3P-AME-DO3P
1
h
2 h
4 h
Blood
3.12 ± 0.65
2.1 ± 0.46
1.13 ± 0.52
2.5 ± 0.43
1.96 ± 0.41
7.73 ± 2.65
1.49 ± 0.35
2.5 ± 0.38
1.16 ± 0.15
8.07 ± 1.79
2.58
2.03 ± 0.25
1.03 ± 0.55
0.98 ± 0.19
2.44 ± 0.22
1.4 ± 0.12
5.04 ± 2.32
1.3 ± 0.09
2.16 ± 0.33
0.59 ± 0.03
10.68 ± 0.46
5.26
1.32 ± 0.27
0.87 ± 0.13
0.80 ± 0.07
1.83 ± 0.06
1.32 ± 0.02
3.6 ± 0.63
1.02 ± 0.03
2 ± 0.12
0.54 ± 0.02
10.13 ± 1.02
7.67
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Heart
Lungs
Liver
Spleen
Kidney
Stomach
Intestine
Muscle
Bone
Acknowledgement
We are thankful to the Director, Institute of Nuclear Medicine and
Allied Sciences, Delhi for providing the necessary facilities. This project
is funded by Defence Research and Development Organization, Ministry
of Defence, India, under project TD-16-17/INM-321.
Bone/Blood
Bone/Muscle
6.95
18.10
18.75
2
3
Appendix A. Supplementary data
bone uptake at 4 h and showed ~ 5.21 %ID/g bone uptake. The bone
6
8
18
uptake of Ga-DO3P-AME-DO3P is inferior to widely used F-NaF
1
8
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2020.104185.
(
~24% ID/g in 60 min),[31] however, F is limited to the need for the
expensive cyclotron. The other phosphonate based agents reported in
6
8
the literature display varied bone uptake capability like Ga-PhenA-
6
8
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