DOI 10.1515/ract-2013-2158 | Radiochim.
Acta 2014; 102(6): 523534
Sudipta Chakraborty, Rubel Chakravarty, Haladhar Dev Sarma, Maroor Raghavan
Ambikalmajan Pillai, and Ashutosh Dash *
Utilization of a novel electrochemical 90Sr/90Y
generator for the preparation of 90Y-labeled RGD
peptide dimer in clinically relevant dose
Abstract: The work reported in this paper provides a sys-
tematic study towards the development of an optimized
strategy for preparation of a clinically relevant dose
of 90 Y -labeled dimeric RGD peptide derivative, DOTA-
E[c(RGDfK)]2 [DOTA-(RGD)2 ] for in vivo targeted ther-
apy utilizing 90 Y obtained from a novel electrochemi-
cal 90 Sr/90 Y generator. The performance of the gener-
ator was evaluated to ensure its suitability for provid-
ing 90 Y in adequate quantity and purity required for for-
mulation of clinically relevant dose for PRRT. 90 Y -DOTA-
(RGD)2 was synthesized in high yield (86.2 2.5%) and ra-
diochemical purity (98.4 0.5%) using clinically relevant
dose (3.8 GBq) of 90 Y . In vitro stability studies revealed
that the radiolabeled conjugate retained its radiochemi-
cal purity in normal saline and human serum. Preliminary
biodistribution studies carried out in C57/BL6 mice bear-
ing melanoma tumors showed that the preparation exhib-
ited significant tumor uptake (5.30 0.78% of injected ac-
tivity at 30 min post-injection) with good tumor to back-
ground ratio. The optimized radiolabeling protocol seems
to be an attractive strategy which is largely viewed as
a springboard to realize scope of developing 90 Y labeled
cyclic RGD peptides for targeted therapy of tumors over-
expressing integrin- 3 receptors.
Keywords: Integrin 3 , Targeted therapy, RGD peptide
dimer, Electrochemical 90 Sr/90 Y generator, 90 Y -DOTA-
(RGD)2 .
||
*Corresponding Author: Ashutosh Dash , Radiopharmaceuticals
Division, Bhabha Atomic Research Centre, Mumbai, India 400
085, India, e-mail: adash@barc.gov.in
Sudipta Chakraborty, Rubel Chakravarty, Maroor Raghavan Am-
bikalmajan Pillai: Radiopharmaceuticals Division, Bhabha Atomic
Research Centre, Mumbai, India 400 085, India
Haladhar Dev Sarma: Radiation Biology and Health Sciences Divi-
sion, Bhabha Atomic Research Centre, Mumbai 400 085, India
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1 Introduction
In recent years, targeted radiotherapy using emitting
radionuclides has not only attracted tremendous attention
but also witnessed unprecedented growth of research and
applications. Among the radionuclides used for therapy,
90
Y is of great practical interest due to its attractive nu-
clear properties such as a short half life of 64.1 h, emis-
sion of high energy radiations (max = 2.28 MeV) and
absence of gamma radiations. The half-life of 90 Y (64.1 h)
is short enough to achieve a critical dose rate and at the
same time is long enough to allow the radiopharmaceu-
tical to be manufactured and delivered for clinical use.
The well-defined coordination chemistry of yttrium in +3
oxidation state provides the scope for complexation with
a plethora of chelators, conjugated with peptides or other
macromolecules used in therapy. The past few decades
have witnessed unprecedented endeavors for developing
90
Y radiopharmaceuticals, and several established 90 Y -
based radiopharmaceuticals are currently in use for treat-
ing various diseases, mainly cancer [1].
While the favorable nuclear characteristics and
amenable solution chemistry of 90 Y hold significant
promise for the preparation of various receptor-based
therapeutic radiopharmaceuticals, the need to achieve
high specific activity of the radiolabeled agent neces-
sitates the use of 90 Y of the highest specific activity
available, as can be obtained from 90 Sr/90 Y generator
system. While 90 Y radiopharmacy lives at the interface
between many disciplines, it is inextricably linked to,
and dependent on, 90 Sr/90 Y generators, which arguably
represents the cornerstone for its survival and success.
As the parent radionuclide, 90 Sr , has a physical half-life
of 28 years, the life span of a generator hence can
potentially be over 10 years. Spurred by the necessity to
explore the scope of using 90 Y in radiopharmaceutical
settings, several 90 Sr/90 Y generators have been developed
and used over the past several years which are reviewed
elsewhere [1, 2]. In order to circumvent the limitations of
the conventional approaches for separation of 90 Y from
524 | S Chakraborty et al., 90 Y-labeled RGD dimer using electrochemical 90 Sr/90 Y generator
90 90
Sr , we have successfully developed a simple Sr/90 Y
generator exploiting the potential of electrochemical
separation approach [3, 4]. This innovative approach of
producing 90 Y with its own specific strengths represents
a new paradigm, and a thorough assessment of its poten-
tial for the preparation of radiopharmaceutical was hence
considered worthwhile investigating.
It is well known that tumors produce many angiogenic
factors, which could activate endothelial cells and induce
endothelial proliferation, migration, and new vessel for-
mation [57]. The angiogenic process is regulated by cell
adhesion receptors. Among the various biochemical fac-
tors regulating angiogenesis, integrin 3 plays a signif-
icant role in cellular adhesion to the extracellular matrix
proteins and subsequent migration of cells [710]. Inte-
grin 3 , up regulated in the activated endothelial cells
of the neovasculature as well as in a variety of growing
tumor cells, is therefore identified as one of the molecu-
lar targets for early diagnosis as well as therapy of rapidly
growing and metastatic tumors [1017]. Over the last two
decades extensive studies have shown that integrin 3
function can be effectively inhibited by small peptides that
posses the Arg-Gly-Asp (RGD) tripeptide sequence [18
20] . Incorporation of this sequence in cyclic penta- and
hexapeptides containing D-amino acids, in a systematic
manner resulted in highly potent and selective inhibitor
of inegrins [1116]. Based on this concept, a large number
of radiolabeled cylic RGD peptides have been developed
and evaluated for their potential as radiotracers as angio-
genesis markers for non-invasive imaging of tumor growth
and metastases in animal models as well as in human pa-
tients [1417, 2135]. In contrast to this, reports on the uti-
lization of radiolabeled RGD peptides in receptor targeted
radionuclide therapy are typically rare till date. Only in
the last couple of years, a few reports have described the
use of tumor targeting properties of radiolabeled RGD pep-
tides beyond diagnostic applications and therapy monitor-
ing to targeted tumor therapy using therapeutic radionu-
clides viz. 90 Y and 177 Lu [3639] .
It is already well documented that with increase in
peptide multiplicity there is an increase in radiotracer
tumor uptake and retention of radiolabeled RGD multi-
mers [14, 21, 2527, 3032, 37, 4042]. This trend is ob-
served from monomer to tetramer. However, a steady in-
crease of uptake in other non-target organs e.g. liver, in-
testine and kidneys is also reported. A comparison of bio-
logical behavior of radiolabeled RGD monomer, dimer and
tetramer revealed that dimers exibit considerable tumor
uptake with highest target/non-target ratio and are suit-
able candidates for tumor imaging and therapy [14, 21,
25, 32, 37, 4042]. Liu et al. have recently reported that
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90
Y -labeled dimeric RGD peptide derivative viz. 90 Y -DOTA-
PEG4 -E[PEG4 -c(RGDfK)]2 , effectively inhibited the growth
of U87MG tumors in a nude mouse model along with sig-
nificantly low radiotoxicity in normal organs [37]. Consid-
ering the future potential of 90 Y -labeled dimeric RGD pep-
tide derivatives towards its clinical utilization as a magic
bullet, developing an optimized protocol for the prepara-
tion of such an agent in clinically relevant dose (3.7 GBq)
of 90 Y was felt pertinent.
We report herein the long term studies of the elec-
trochemical 90 Sr/90 Y generator to evaluate its perfor-
mance and development of an optimized strategy for the
preparation of 90 Y -labeled dimeric RGD peptide derivative
DOTA-E[c(RGDfK)]2 (Figure 1) in clinically relevant dose
of 3.7 GBq using 90 Y obtained from the aforementioned
generator. We also describe an overview of radiolabeling
procedure and assessment of the quality of the 90 Y -DOTA-
(RGD)2 conjugate for therapeutic use.
2 Experimental
2.1 Materials and methods
The RGD peptide derivative viz. DOTA-E[c(RGDfK)]2 (DOTA-
RGD2 , > 99% chemically pure) (Figure 1) was custom syn-
thesized by ABX Advanced Biochemical Compounds, Ger-
many. Gentisic acid (2,5-dihydroxybenzoic acid) was ob-
tained from Aldrich Chemical Company, USA. Suprapure
hydrochloric acid was purchased from Merck, Germany.
MilliQ water (resistivity 18.2 M cm) was used for all the
studies. All other chemicals used in the experiments were
of Analytical Reagents (AR) grade and supplied by reputed
chemical manufacturers. Carrier yttrium chloride solution
was prepared by dissolving a known amount of natural
Y2 O3 (specroscopic grade, mononuclidic in 89 Y) in 0.1 M
suprapure HCl.
Assay of 90 Y activity obtained from the generator was
carried out by using a liquid scintillation counter (Model
Tricarb 2100TR Packard Instrument Co., USA). Radionu-
clidic purity of 90 Y was ascertained by following the ex-
traction paper chromatography (EPC) technique [43]. The
procedure is based on the selective retention of 90 Y by
bis(2-ethyl hexyl)phosphonic acid (HDEHP), the chelate
impregnated at the point of spotting of the paper chro-
matography strip (Whatman 3 MM, 12 1 cm). Briefly,
A 5 L aliquot of 90 Y -acetate test solution containing
0.185 MBq (5 Ci) of 90 Y was applied on the paper which
was developed in normal saline. The paper strip was dried,
cut into 1 cm segments and the radioactivity associated
with each segment were measured by liquid scintillation
 S Chakraborty et al., 90 Y-labeled RGD dimer using electrochemical 90 Sr/90 Y generator | 525

O NH NH O
O O
N N NH2 H2N N N
H H H H
HO NH HN O O NH HN OH
O O
O N O
O NH HN N NH HN O
H H
O NH
O CO2H O
N N

N N
CO2H CO2H

Fig. 1: Structure of DOTA-E[c(RGDfK)]2 (DOTA-RGD2 ).

counting. The 90 Sr activity present at the solvent front was
then compared to the total applied activity to obtain the
radionuclidic impurity levels. Radiochemical purity was
determined by paper chromatography using saline as the
mobile phase and by paper electrophoresis [44]. Chemi-
cal purity was determined by Inductively Coupled Plasma
Atomic Emission Spectroscopy (ICP-ES JY-238, Emission
Horiba Group, France) analysis of the decayed sample.
All other radioactivity measurements were carried out
by using well type NaI(Tl) scintillation counter (Mucha,
Raytest, Germany) utilizing the bremstrahlung photons.
The high performance liquid chromatography (HPLC) sys-
tem equipped with a PU 1575 UV/VIS detector was ob-
tained from JASCO (PU 1580), Japan. A well-type NaI(Tl)
scintillation detector (Mucha, Raytest, Germany) was cou-
pled to the system for radioactivity measurements in the
eluate. All the solvents used for HPLC analyses were
of HPLC grade and purchased from Merck, India, de-
gassed and filtered prior to use. Sep-Pak C18 1 cc Vac
cartriges containing hydrophobic, reverse-phase slilica-
based bonded phase were used for the purification of ra-
diolabeled conjugate and were obtained from Waters, Ire-
land.
Female C57/BL6 mice (6 8 weeks age) bearing
melanoma tumors were used for evaluating the tu-
mor avidity of synthesized radiolabeled conjugate. The
melanoma cell line (ATCC-CRL-6475), used for inocula-
tion of the tumors, was purchased from National Center
for Cell Sciences, India. The animals were bred and reared
in the laboratory animal facility of our Institute under
standard management practice. Radioactivity measure-
ments associated with the animal studies were carried
out using a flat-type NaI(Tl) scintillation detector. All the
animal experiments reported in the article were carried
out in strict compliance with the relevant national laws
relating to the conduct of animal experimentation.
2.2 Electrochemical 90 Sr/90 Y generator
Yttrium-90 used for the present study was obtained
from an electrochemical 90 Sr/90 Y generator developed in-
house [4]. In brief, a 0.001 M HNO3 solution containing
5.55 GBq (150 mCi) of 90 Sr in equilibrium with 90 Y was
used as an electrolyte. The electrochemical separation
process involved two electrolysis cycles the first cycle
for separation and the second cycle for purification of 90 Y .
The first cycle involved electrolysis of a mixture of 90 Sr and
90
Y in nitrate form at pH 3 at a potential of 2.5 V, us-
ing platinum electrodes. The cathode containing 90 Y de-
posit was removed and transferred to a new electrolysis
bath containing 3 mM HNO3 as the electrolyte and an-
other platinum electrode. The polarity of the electrodes
was reversed, thereby using the cathode from the first elec-
trolysis cycle containing 90 Y as the anode and the elec-
trolysis process was repeated. Upon electrolysis, 90 Y was
leached and deposited on the fresh cathode, which was
removed and dipped in 0.1 N HCl to obtain 90 Y Cl3 , which
was subsequently used for radiolabeling.
2.3 Quality control of 90 Y
Prior to radiolabeling, radionuclidic purity of 90 Y was
evaluated following the extraction paper chromatography
(EPC) technique [43]. In order to estimate the levels of trace
metal ions (Zr4+ , Fe3+ , Cu2+ and Zn2+ ) in the 90 Y Cl3 eluted
from the generator, few batches of the 90 Y Cl3 were allowed
to decay for two months and were subjected to inductively
coupled plasma atomic emission spectroscopy (ICP- AES)
analysis.

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526 | S Chakraborty et al., 90 Y-labeled RGD dimer using electrochemical 90 Sr/90 Y generator
2.4 Preparation of 90 Y-DOTA-(RGD)2
radiotracer
90
For the preparation of Y labeled DOTA-(RGD)2 tracer,
a stock solution of the peptide conjugate was prepared in
MilliQ water with a concentration of 1 mg/mL. A 25 L
aliquot of this solution containing 25 g (14.7 nM) of the
ligand was taken and 175 L of 0.1 M ammonium ac-
etate buffer of pH 5.5 and 50 L aliquot of 90 Y Cl3 solution
in 0.1 M HCl (37 MBq) obtained from the generator was
added. The reaction mixture was incubated at 90 C for
30 min after adjusting the pH to 4. Various parameters,
such as, amount of ligand used (2 50 g), pH of the reac-
tion mixture (2-8), incubation time (10 60 min) and tem-
perature (room temperature and 90 C) were varied in or-
der to arrive at the optimized protocol for obtaining maxi-
mum complexation as mentioned above.
2.5 Determination of yield and
radiochemical purity
The complexation yield and radiochemical purity of the
synthesized radiotracer was determined by HPLC. HPLC
was carried out using a dual pump HPLC unit with a C-18
reversed phase HiQ-Sil (5 M, 25 cm 0.46 cm) column.
The elution was monitored both by detecting UV signals
at 270 nm as well as by radioactivity signal using NaI(Tl)
detector. Water (A) and acetonitrile (B) mixtures with 0.1%
trifluoroacetic acid were used as the mobile phase and the
following gradient elution technique was adopted for the
separation (0 5 min 95% A, 5 15 min 95% A to 5% A,
15 20 min 5% A, 20 25 min 5% A to 95% A, 25 30 min
95% A). Flow rate was maintained at 1 mL/min.
2.6 Determination of octanol/water
partition coefficients
The octanol/water partion coeffient was detemined using
the following protocol. A 25 L aliquot of the 90 Y -DOTA-
(RGD)2 complex prepared under optimized conditions was
diluted to 1 mL volume using normal saline. To this solu-
tion 1 mL of n-octanol was added and the mixture stirred
vigorously for 10 min. Subsequently, the mixture was
centrifuged at a speed of 3000 rpm for 5 min. Aliquots
were withdrawn from both water and n-octanol layer and
counted in NaI(Tl) counter. The Log P value was deter-
mined from these data and reported as an average of three
independent measurements.
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2.7 Maximization of [L]/[M] ratio
Tracer level experiments were carried out to achieve
maximum complexation yield of 90 Y -DOTA-(RGD)2 us-
ing the minimum possible [L]/[M] ratio. For this, 25 g
(14.7 nM) of the peptide conjugate (already optimized lig-
and amount) was allowed to react with different amounts
of carrier yttrium ranging from 0.1 g (1.1 nM) to 1.0 g
(11.2 nM) under the same reaction conditions described
earlier. The complexation yields were determined follow-
ing the technique described earlier.
2.8 Preparation of 90 Y-DOTA-(RGD)2 in
clinically relevant dose
Clinically relevant dose of 90 Y -DOTA-(RGD)2 uing 4.5 GBq
of 90 Y was prepared in the following way. To 500 L of
0.1 M ammonium acetate buffer (pH 5.5), were added
25 L of DOTA-(RGD)2 solution in MilliQ water (1 g/L
concentration, 25 g, 14.7 nM peptide conjugate) and
200 L of 90 Y Cl3 solution (4.5 GBq of 90 Y ) in 0.01 M HCl.
The pH of the resultant mixture was adjusted to 44.5
by addition of 0.01 M NaOH solution. Subsequently, the
reaction mixture was incubated at 90 C for 30 min.
After the completion of the reaction the mixture was al-
lowed to cool till it reached room temperature, a small
aliquot was withdrawn and complexation yield was deter-
mined. Subsequently, the radiolabeled conjugate was pu-
rified using SepPak catridge. For this, the reaction mixture
was loaded into the Sep-Pak catridge pre-conditioned by
passing 2 mL of 0.1 M ammonium acetate buffer through
it. Uncomplexed 90 Y +3 was removed by passing 2 mL
of 0.1 M ammonium acetate buffer. 90 Y -labeled DOTA-
(RGD)2 was eluted in 1 mL of ethanol. Finally, ethanol was
evaporated off; the purified radioconjugate was reconsti-
tuted in 1 mL of normal saline containing 1 mg of gen-
tisic acid and subjected to radiochemical purity analysis
by HPLC.
2.9 In vitro stability studies
The stability of 90 Y -DOTA-(RGD)2 prepared in 4.5 GBq
dose was studied in normal saline as well as in hu-
man serum. Purified 90 Y -DOTA-(RGD)2 solution in normal
saline containing 1 mg of gentisic acid was stored at room
temperature up to 7 d. The radiochemical purity of the
preparation was determined at different time intervals us-
ing HPLC technique. In order to determine the in vitro sta-
bility of the complexes in human serum, 100 L aliquot
 S Chakraborty et al., 90 Y-labeled RGD dimer using electrochemical 90 Sr/90 Y generator
of the purified preparation was added to 900 L of freshly
separated human serum incubated at 37 C upto a period
of 72 h. Aliquots were withdrawn at different time inter-
vals, ethanol was added to precipitate the serum protein,
centrifuged and the supernatant carefully separated. The
percentage of activity of the radiotracer precipitated with
serum protein was determined by measuring the activity of
the precipitate as well as the supernatant. The supernatant
was then analyzed by HPLC to ascertain radiochemical pu-
rity of 90 Y -DOTA-(RGD)2 .
2.10 Biodistribution studies
Biological behavior of 90 Y -DOTA-(RGD)2 was studied in
C57/BL6 mouse bearing melanoma tumor. Melanoma tu-
mors were developed by injecting 1 106 melanoma
cells (ATCC-CRL-6475) suspended in 200 L of PBS sub-
cutaneously into the right thigh of each C57/BL6 mice
weighing 20 25 g. The animals were observed for visibil-
ity of tumors and subsequently allowed to grow for about
2 weeks to attain a tumor mass of 0.2 0.4 g. An aliquot
of 90 Y -DOTA-(RGD)2 prepared in 3.8 GBq dose was di-
luted in normal saline. Approximately, 7.4 MBq of radio-
tracer in 100 L solution was injected into each animal
through a lateral tail vein. The animals were sacrificed by
cardiac puncture post-anesthesia at 30 min, 3 h, 24 h and
72 h post-injection (p.i.). Four animals were used in each
time point. Various organs, tissue and tumors were excised
following sacrifice, washed with normal saline, dried,
weighed using analytical balance and the radioactivity as-
sociated with each organ and tissue was determined us-
ing a flat type NaI(Tl) counter. The percent injected activity
(%ID) in various organs, tissue and tumor were calculated
from the above data and expressed as percentage injected
activity per gram (%ID/g) of organ/tissue. The activity ex-
creted was indirectly determined from the biodistribution
pattern and not by measuring the activity in the urine and
feces. The %ID measured in all the organs were summed
up. Percentage excretion was obtained from the difference
between total injected activity (ID) and the sum of %ID ac-
counted for in all the organs. The total uptake in blood,
bone and muscles were calculated by assuming that 7%,
10% and 40% of the body weight are constituted by these
organs/tissues, respectively.
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| 527
3 Results
3.1 Evaluation of electrochemical 90 Sr/90 Y
generator
In light of the explicit need to realize the utility of elec-
trochemical 90 Sr/90 Y generator to provide 90 Y for radiola-
beling, a 5.55 GBq (150 mCi) generator was used and its
performance for a period of 1 year was evaluated. Table 1
depicts the performance of the generator in 10 different
random batches. As seen from the result, the electrochem-
ical 90 Sr/90 Y generator was found to be effective in pro-
viding 4.44 GBq (120 mCi) of no-carrier-added (NCA)
grade 90 Y per batch. No significant difference in 90 Y yields
was observed from batch to batch and the 90 Y yield was al-
ways > 80% which indicates the highly reproducible per-
formance of this generator. The breakthrough is expressed
as a percentage of the 90 Sr activity in the separated 90 Y
at time of separation. Noticeably, the 90 Sr/90 Y ratio of the
separated product was observed to be < 8 107 which is
well within the pharmacopia limits (2 105 ) as estimated
by extraction paper chromatography [45].
The levels of trace metal ions, namely Zr4+ , Fe3+ , Cu2+
and Zn2+ , in the 90 Y obtained from the generator as ana-
lyzed by ICP-AES were found to be below detectable limit
(0.1 mg/L, 0.1 ppm for all the metal ions), thereby con-
firming their absence in the generator produced 90 Y . While
ICP-AES method of assessing metallic impurities is a post-
facto confirmation, this exercise was crucial mainly to reaf-
firm that 90 Y obtained from the generator is free from
metallic impurities. From the perspective of the perfor-
mance evaluation, the generator was able to provide re-
producible yields of 90 Y activity with minimal metallic im-
purities and 90 Sr breakthrough remain within permissi-
ble level. It is important to note that, during the process
demonstration run for a period of 2 years, the yield and
purity of 90 Y remained unaltered.
Having successfully completed the performance eval-
uation of the electrochemical 90 Sr/90 Y generator, our sub-
sequent efforts were directed towards realizing the utility
of 90 Y obtained from this generator for the radiolabeling
of DOTA-(RGD)2 conjugate using a clinically relevant dose.
3.2 Characterization of 90 Y labeled
DOTA-(RGD)2 conjugate
The 90 Y labeled DOTA-(RGD)2 complex was characterized
by HPLC. It was observed that, the 90 Y labeled conjugate
eluted as a single peak with a retention time of 17.4 min
while uncomplexed 90 Y Cl3 eluted from the column within
528 | S Chakraborty et al., 90 Y-labeled RGD dimer using electrochemical 90 Sr/90 Y generator

Table 1: Batch performance of the electrochemical 90 Sr/90 Y generator.

Batch Amount of Expected Y90

% 90
Sr/90 Y Level of Zr4+ ,
90 90
No Sr in the Y obtained Yield ratio Fe3+ , Cu2+ and
feed GBq (mCi) GBq (mCi) Zn2+ impurities
GBq (mCi) (ppm)

1 4.55 (123) 83.6 7 107 < 0.1 ppm

2 4.73 (128) 87.1 5 107 < 0.1 ppm
3 4.36 (118) 80.2 9 107 < 0.1 ppm
4 4.44 (120) 81.6 2 107 < 0.1 ppm
5 5.55 (150) 5.44 (147) 4.62 (125) 85.0 1 106 < 0.1 ppm
6 4.74 (128) 87.1 9 107 < 0.1 ppm
7 4.51 (122) 82.9 8 107 < 0.1 ppm
8 4.36 (118) 80.2 1 107 < 0.1 ppm
9 4.70 (127) 86.4 7 107 < 0.1 ppm
10 4.44 (120) 81.6 8 107 < 0.1 ppm

Time allowed for growth of 90 Y = 15 d.

3.0 min. The complexation yield and the radiochemical
purity of the complex could therefore be determined from
the HPLC studies.
3.3 Optimization studies
In order to determine the optimum ligand concentration
required for obtaining 90 Y labeled DOTA-(RGD)2 with near-
quantitative yield and purity, the amount of DOTA-(RGD)2
was varied from 2 g (1.2 nM) to 50 g (29.3 nM) in 250 L
reaction volume using 37 MBq of 90 Y activity (1.8 ng,
20.5 pM of Y). It was observed that a complexation yield of
> 98% could be obtained when 25 g (14.6 nM) of DOTA-
(RGD)2 was used. The effect of the amount of peptide con-
jugate on the complexation yield of 90 Y -DOTA-(RGD)2 is
100
shown in Figure 2. Studies on the effect of pH of the re-
action mixture on complexation yield within a pH range
of 2-8 indicated that the yield was maximal at pH 4.5.
Complexation studies beyond the above mentioned pH
range was not attempted as drastic reaction conditions
could damage the integrity of conjugate in addition to the
possibility of formation of 90 Y -hydroxide at higher pH. In
order to ascertain the optimum reaction condition, reac-
tions were carried out by incubating the reaction mixture
at room temperature and 90 C for different time periods
(5, 10, 20, 30 and 60 min) at already optimized ligand con-
centation and pH. It was observed that, > 98% complexa-
tion was achieved within 30 min when the reaction mix-
ture was incubated at 90 C. On the other hand, 82%
complexation could be achieved when the reaction mix-
ture was incubated for 60 min at room temperature. There-
fore, 30 min incubation at 90 C was considered as the op-
timum condition for radiolabeling.

80 3.4 Octanol/water partition coefficient

% Complexation Yield

The log P value of 90 Y -DOTA-(RGD)2 radioconjugate was

60 found to be 2.67 0.08 ( = 6) indicating that the com-
plex is highly hydrophilic in nature.
40

3.5 Maximization of the specific activity of

20 90
0 5 10 15 20 25 30 Y-DOTA-(RGD)2
[DOTA-RGD2] (nM)
Bearing in mind the significance of specific activity of
Fig. 2: Effect of the amount of DOTA-RGD2 on the complexation yield radiolabeled receptor specific biomolecules for use in in
of 90 Y-DOTA-RGD2 . vivo targeted therapy, such as radiolabeled peptides for

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 S Chakraborty et al., 90 Y-labeled RGD dimer using electrochemical 90 Sr/90 Y generator | 529

Table 2: Complexation yield of 90 Y-DOTA-RGD2 using different plexation, the ligand concentration is kept constant at this
amounts of carrier yttrium. value and complexations were carried out using different
amounts of yttrium ranging from 0.1 g (1.1 nM) to 1.0 g
Amount of Amount of [L]/[M] % Complexation (11.2 nM) under the same reaction conditions described
DOTA-RGD2 [Y] yield earlier. The complexation yields obtained at different con-
NCA 90 Y (20.5 pM) 717.3 98.2 0.6 centrations of yttrium are given in Table 2. It is evident
25 g 0.1 g (1.1 nM) 13.4 98.9 0.4 from the results that a radiolabeling yield of >98% could
(14.7 nM) 0.2 g (2.2 nM) 6.7 98.6 0.3 be achieved using 25 g (14.7 nM) peptide conjugate and
0.5 g (5.6 nM) 2.6 98.0 1.1
1.0 g (11.2 nM) 1.3 78.3 1.5
0.5 g (5.6 nM) of yttrium. Using NCA 90 Y , 0.5 g of yt-
trium carrier corresponds to 10 GBq (271.6 mCi) of 90 Y
activity. Hence the present study demonstrates that a clin-
use PRRT, efforts were directed towards the preparation of ically relevant dose of 3.7 GBq (100 mCi) of 90 Y -DOTA-
90
Y -DOTA-(RGD)2 with maximum possible specific activity (RGD)2 could be synthesized using 25 g of the peptide
without compromising the yield and stability. For this, the conjugate.
[L]/[M] ratio used for a typical labeling procedure needs
to be minimum in order that amount of unlabeled peptide
which would otherwise bind also with the target recep- 3.6 Clinically relevant dose of
tors will be minimum. The [L]/[M] ratio can be minimized 90
Y-DOTA-RGD2
by keeping the concentration of the peptide constant and
gradually increasing the amount of radiometal in the re- Attempts to prepare 90 Y -DOTA-(RGD)2 using 4.5 GBq of
90
action mixture. Since 25 g of the peptide was found to be Y activity following the method described in the exper-
the optimum amount required for obtaining > 98% com- imental section yielded the radioconjugate with a radio-

Fig. 3: Typical HPLC radiochromatograms of 90 Y-DOTA-RGD2 immediately after preparation (A) and after 6 d storage at room temprerature in
normal saline (B).

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530 | S Chakraborty et al., 90 Y-labeled RGD dimer using electrochemical 90 Sr/90 Y generator

100 post-preparation studied. The HPLC chromatograms of

the product recorded at different time intervals post-
98 preparation did not show any appreciable degradation
% Radiochemical Purity

of the radiolabeled conjugate. A tpyical HPLC radiochro-

96 matogram of the radiolabeled conjugate recorded 7 d post-
preparation is given in Figure 3(B) showing that the con-
94 jugate retained its radiochemical purity to the extent of
97.3%. The variation of radiochemical purity of 90 Y -DOTA-
92 (RGD)2 when stored in human serum at 37 C for different
durations is shown in Figure 4. It is evient from the figure
90 that the radiolabeled conjugate retained its radiochemical
0 16 32 48 64 80 purity to the extent of > 95% in human serum at 37 C up
Incubation time (h) to 72 h post-preparation studied. Also, it was observed that
97% of the activity of the radiotracer mixed with human
Fig. 4: Variation of radiochemical purity of 90 Y-DOTA-(RGD)2 when
stored in human serum at 37 C for different durations.
serum remained in the supernatant after the precipitation
of serum protein using ethanol.

chemical putity of 86.2 2.5% ( = 3). However, 3.8 GBq

(102.7 mCi) of purified 90 Y -DOTA-(RGD)2 was recovered
post-purification using SepPak catridge having radio-
chemical purity of 98.6 0.6% ( = 3). A typical HPLC
radiochromatogram of the radiolabeled conjugate post-
purification is shown in Figure 3(A). The specific activity
the radiolabeled conjugate was 260 GBq/M.
3.7 In vitro stability
The 90 Y -DOTA-(RGD)2 formulation with clinically rele-
vant dose of 3.8 GBq of 90 Y was found to be stable in
vitro in normal saline at room temperature up to 7 d
3.8 Biodistribution and imaging studies
The uptake of 90 Y -DOTA-(RGD)2 conjugate in different
organs/tissue of C57/BL6 mice bearing melanoma tu-
mors expressed as percentage injected activity per gram
of organ/tissue (%ID/g) at different post-injection (p.i.)
times is shown in Table 3. The results of the biodistri-
bution studies revealed significant tumor uptake within
30 min p.i. (5.30 0.78% D/g). Accumulation of activity
was also observed in various non-target organs such as
liver, intestine kidney etc. at 30 min p.i. However, with
the progress of time, the accumulated activity in the non-
target organs gradually cleared through renal route. The

Table 3: Biodistribution pattern of 90 Y-DOTA-RGD2 conjugate in C 57/BL6 mice bearing melanoma tumors.

Organ % Injected activity per gram (%D/g) of organ/tissue

30 min 3h 24 h 72 h

Blood 2.13 (0.18) 0.55 (0.12) 0.13 (0.03) 0.03 (0.03)

Liver 4.37 (0.66) 3.31 (0.85) 2.72 (0.38) 1.25 (0.13)
GIT 1.37 (0.10) 0.58 (0.24) 0.27 (0.08) 0.10 (0.03)
Kidney 8.37 (0.79) 5.26 (1.01) 4.23 (0.79) 2.12 (0.44)
Stomach 0.24 (0.11) 0.20 (0.04) 0.15 (0.03) 0.13 (0.04)
Heart 1.42 (0.15) 0.47 (0.11) 0.25 (0.10) 0.00 (0.00)
Lungs 2.43 (0.32) 2.01 (0.35) 1.36 (0.28) 0.92 (0.17)
Skeleton 0.22 (0.05) 0.20 (0.07) 0.10 (0.04) 0.05 (0.02)
Muscle 0.38 (0.12) 0.21 (0.06) 0.25 (0.06) 0.11 (0.02)
Spleen 3.82 (0.56) 2.49 (0.23) 1.32 (0.37) 0.41 (0.07)
Tumor 5.30 (0.78) 4.03 (0.69) 3.32 (0.41) 1.93 (0.51)
Excretion# 82.18 (4.31) 84.72 (3.00) 86.19 (1.23) 94.61 (2.22)
Figures in the parentheses represent standard deviations.
At every time point 4 animals have been used.
#
Excretion has been calculated by subtracting the activity accounted in all the organs from the total activity injected.

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 S Chakraborty et al., 90 Y-labeled RGD dimer using electrochemical 90 Sr/90 Y generator | 531

2.0
80

1.6
60

Tumor / Liver
1.2
Tumor / Blood

40
0.8

20
0.4

0 0.0

(A) (B)
1.5

25

1.0 20
Tumor / Kidney

Tumor / Muscle
15

0.5 10

0.0 0
30 min 3h 24 h 72 h
Time p.i.

(C) (D)

Fig. 5: Tumor to background ratio of 90 Y-DOTA-(RGD)2 at different time points post-injection: tumor/blood (A), tumor/liver (B), tumor/kidney
(C) and tumor/muscle (D).

tumor to background ratio exhibited by the radiolabeled
conjugate at different time points post-injection with re-
spect to the major organs/tissue are shown in Figure 5
(AC). It is evident that the conjugate exhibited favourable
tumor to blood and tumor to muscle ratio. The tumor to
liver ratio, although not very high, is reasonably good for
a therapeutic agent. However, the retention of activity in
the kidneys up to 24 h p.i. (4.23 0.79% D) led to not so
encouraging tumor to kidney ratio (Figure 5C). The radi-
olabeled conjugate exhibited predominant urinary excre-
tion, as 82.18 4.31% D was observed to be cleared via re-
nal pathway within 30 min p.i. The initially accumulated
activity in the melanoma tumor was observed to decrease
gradually, although 1.93 0.51% D/g was retained in tu-
mor at 72 h p.i. up to which time the biodistribution stud-
ies were continued.
4 Discussions
The advantages of 90 Sr/90 Y generator system as the ex-
clusive source for availing NCA 90 Y for receptor-mediated
targeted therapy needs hardly to be reiterated. At the
intersection of receptor-mediated radionuclide therapy
and 90 Sr/90 Y generator lays the thriving world of ra-
diolabeled RGD (Arg-Gly-Asp) peptides which can open
the door to uncover numerous exhilarating possibili-
ties to bring major therapeutic breakthroughs in cancer
patients.
With the ever increasing demands of 90 Y for therapy,
the need for a reliable 90 Sr/90 Y generator is imminent.
This investigation underscores the utility of electrochem-
ical 90 Sr/90 Y generator [3, 4] to meet the stated goals and
at the same time addresses the cognitive and contextual
concerns. The payoff will be an increased supply of 90 Y to
spawn research on new radiopharmaceuticals with 90 Y for
therapy. While the use of electrochemical 90 Sr/90 Y gener-
ator system constitutes a positive step in the right direc-
tion to ensure convenient availability 90 Y for radionuclide
therapy, the possible contamination of 90 Sr (1/2 = 28.8 y)
in the separated 90 Y poses a serious risk owing to the ra-
diotoxicity of 90 Sr . In view of this consideration, assessing
the purity of the 90 Y prior to the preparation of the radio-
conjugate for therapy represents a necessity to ensure that
it is well within the acceptable limits. According to the US
Pharmacopeia, 90 Sr/90 Y activity ratio should not exceed
2 105 parts at the time of injection of any 90 Y -based ra-
diopharmaceuticals [45]. In this context, the exquisite sen-
sitivity and specificity of Extraction Paper Chromatogra-

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532 | S Chakraborty et al., 90 Y-labeled RGD dimer using electrochemical 90 Sr/90 Y generator
phy (EPC) technique [43] seemed to be an attractive real
time QC procedure owing to its ability to detect few Bq of
90
Sr in MBq quantities of 90 Y .
The integrin 3 is an adhesion molecule that not
only mediates migration of cells on the extracellular ma-
trix but also acts as a receptor that senses the interac-
tion of cells with the extracellular matrix and activates in-
tracellular signaling pathways. The restricted over expres-
sion of integrin 3 during tumor growth, invasion, and
metastasis could be effectively exploited as molecular tar-
get for the early tumor detection and noninvasive monitor-
ing of tumor metastasis and therapeutic response. An ideal
radiolabeled RGD peptide of clinical importance should
have high affinity and high specificity for integrin 3 and
a good elimination profile [14].
The enormous potential of electrochemical 90 Sr/90 Y
generator as a convenient source of 90 Y for clinical uti-
lization has motivated us to pursue the radiolabeling
of DOTA-(RGD)2 conjugate with 90 Y for its future po-
tential application in PRRT. This investigation has not
only highlighted the optimization of radiolabeling pro-
cedure but also assess the quality of 90 Y -labeled DOTA-
(RGD)2 conjugate in terms of in vitro stability, tumor up-
take, and tumor to back-ground ratio. In light of the
perceived need to maintain the stability of the 90 Y -
DOTA-(RGD)2 conjugate during therapy, evaluating in vitro
stability of the radiolabeled product in human serum
is not only an interesting prospect but also viewed
as necessary. Result of the experimental studies in-
dicated that 90 Y -DOTA-(RGD)2 conjugate remained sta-
ble in human serum at least for 72 h at 37 C after
preparation.
Preliminary biodistribution studies carried out in
C57/BL6 mice bearing melanoma tumors showed that the
preparation exhibited significant tumor uptake, good tu-
mor to background ratio as described in the section 3.8.
However, further improvement on tumor retention and tu-
mor to background, especially tumor to liver and tumor
to kidney ratio would be always advantageous for a PRRT
agent based on high energy emitting radionuclide such
as 90 Y . Recent reports have shown that integrin target-
ing capability as well as clearance of radiolabeled RGD
peptide dimers from normal organs such as liver, kidneys
etc. can be improved significantly when the distance be-
tween the two RGD motifs is increased by introducing
pharmacokinetic modifying linkers namely triglycine (G3 )
and polyethylene glycol (PEG4 ) [21, 25, 27, 3335, 37, 42].
The similar strategy could be adopted in case of 90 Y -DOTA-
(RGD)2 in order to obtain a better PRRT agent with im-
proved tumor uptake, retention and tumor to background
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ratio. Nonetheless, the optimization of the protocol for
prepation of the clinically useful dose of the agent demon-
strated in the present study using DOTA-(RGD)2 as the pep-
tide conjugate could be utilized for any such 90 Y -based
PRRT agents.
The successful preparation of a clinically relevant
dose ( 3.8 GBq) of 90 Y -DOTA-(RGD)2 conjugate with ade-
quate radiochemical purity and in vitro stability using op-
timized procedure is poised to unveil new dimension to
the receptor-mediated radionuclide therapy. The compli-
ance of 90 Sr impurity limit in the 90 Y -DOTA-(RGD)2 conju-
gate as per pharmacopeia guidelines is an essential pre-
requisite from the perspective of patient safety point of
view [4547] and the reported procedure was found to be
effective in achieving this objective. A striking feature of
electrochemical 90 Sr/90 Y generator is its ability to provide
90
Y with negligible concentration of metal ion impurities
such as Zr4+ , Fe3+ , Cu2+ and Zn2+ which offers possibility to
prepare high dose of 90 Y -DOTA-(RGD)2 in adequately high
specific activity ( 260 GBq/M) of the radiolabeled con-
jugate. In view of the extremely low concentration of NCA
90
Y , competing metal ions even at ppb levels act as pseudo
carriers [48, 49], requiring higher DOTA-(RGD)2 concentra-
tions to achieve high radiolabeling yields which in turn
restrict their utility for clinical use. Thus the aptness of
the electrochemical 90 Sr/90 Y generator for obtaining 90 Y
amenable for clinical use is amply demonstrated. The re-
sults of this investigation viewed largely as a springboard
to spur further development on 90 Y labeled cyclic RGD
peptides for targeted therapy of tumors over-expressing in-
tegrin 3 .
5 Conclusion
The objective of long-term utilization of the electrochem-
ical 90 Sr/90 Y generator towards obtaining 90 Y of requi-
site purity and arriving at radiolabeling conditions of
DOTA-(RGD)2 conjugate commensurate with therapeutic
requirements has been successfully achieved. Taking ad-
vantage of the electrochemical 90 Sr/90 Y generator and
the ability of DOTA-(RGD)2 to form stable radioconju-
gate, captivating advances have been uncovered. Based
on the optimized radiolabeling condition, batches of 90 Y -
DOTA-(RGD)2 containing 3.8 GBq of 90 Y have been suc-
cessfully prepared. The data emerging from our studies
suggest that 90 Y -DOTA-(RGD)2 with favorable properties
would be of great utility given the convenience of 90 Y
production as well as radiolabeling. Given the ever in-
creasing uses of DOTA-(RGD)2 in integrin 3 -positive
 S Chakraborty et al., 90 Y-labeled RGD dimer using electrochemical 90 Sr/90 Y generator
tumors, radiopharmaceutical scientists must continue to
use the electrochemical 90 Sr/90 Y generator system for the
development of new 90 Y based therapeutic agents. Our
findings of this study will inspire and stimulate scien-
tists to pursue further studies not only on 90 Y labeled
cyclic RGD peptides but also on other peptides as target-
ing agents.
Acknowledgement: Research at the Bhabha Atomic Re-
search Centre is part of the ongoing activities of the De-
partment of Atomic Energy, India and is fully supported
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