The 177Lu-[tris(1,10-phenanthroline)lutetium(III)] complex
(177Lu-PQ3) was prepared successfully with high radiochemical
purity (> 99%). Lu-177 chloride was obtained by
thermal neutron flux (4 × 1013 n.cm&#8211;2.s&#8211;1) of natural Lu2(NO3)3
sample, dissolved in acidic media. The radiochemical yield
was checked by measuring the radiochemical purity of the
177Lu-PQ complex by ITLC (10 mM DTPA, pH = 5, as mobile
phase). The final complex solution was injected intravenously
into wild-type male rats and bio-distribution of the complex
was checked for up to 48 hours. The dose limiting organs
were shown to be the reticulu-endothelial system. The
bio-distribution of the labelled compounds in tumour-bearing
animals is under investigation.
Nuclear Med Rev 2010; 13, 2: 49&#8211;5

Bahrami-Samani, A.

Ghannadi-Maragheh, M.

Jalilian, Amir R.

Shirvani-Arani, S.

Yousefnia, Hassan

Zolghadri, S.

English

Via Medica Journals

49
Nuclear Medicine Review 2010
Vol. 13, No. 2, pp. 49–54 
Copyright © 2010 Via Medica
ISSN 1506–9680
www.nmr.viamedica.pl
Review
Abstract
The 177Lu-[tris(1,10-phenanthroline)lutetium(III)] complex 
(177Lu-PQ3) was prepared successfully with high radio-
chemical purity (> 99%). Lu-177 chloride was obtained by 
thermal neutron flux (4 × 1013 n.cm–2.s–1) of natural Lu2(NO3)3 
sample, dissolved in acidic media. The radiochemical yield 
was checked by measuring the radiochemical purity of the 
177Lu-PQ complex by ITLC (10 mM DTPA, pH = 5, as mobile 
phase). The final complex solution was injected intravenously 
into wild-type male rats and bio-distribution of the complex 
was checked for up to 48 hours. The dose limiting organs 
were shown to be the reticulu-endothelial system. The 
bio-distribution of the labelled compounds in tumour-bearing 
animals is under investigation.
Key words: lutetium-177, 1,10-phenanthroquinone, 
biodistribution 
Nuclear Med Rev 2010; 13, 2: 49–54
Preparation and quality control 
of 177Lu-[tris(1,10-phenanthroline) 
lutetium(III)] complex for therapy
Correspondence to: Amir R. Jalilian
Radiopharmaceutical Research and Development Lab (RRDL)
Nuclear Science and Technology Research Institute (NSTRI)
Tehran, Iran, Postal code: 14155–1339
Tel:++98-21-88221103, fax:++98-21-88221105
e-mail:ajalilian@nrcam.org
Introduction
[Tris(1,10-phenanthroline)lanthanum(III)] (La-PQ3) has been 
prepared previously and the rigid planar 1,10-phenanthroline 
(PQ) molecule has demonstrated distinct effects on in vitro 
cultured cells. The complex has also been shown to stop DNA 
synthesis in CCRF-CEM and Ehrlich ascites cells leading to 
a cell cycle arrest in G0/G1 [1, 2], based on the metal chelating 
ability of PQ [3, 4], several metal ions including copper, ruthe-
nium, and cobalt, has been shown to enhance the anticancer 
activity of PQ [5–7]. 
On the other hand, several complexes of vanadium with PQ 
derivatives have been shown to demonstrate an apoptotic ef-
fect in vivo and in vitro [8–10]. Recently, La-PQ3 demonstrated 
anticancer activity via potent induction of cell cycle arrest and/or 
apoptosis and has promising in vivo anticancer activity against 
a human colon cancer xenograft, suggesting La-PQ3 as a new 
anticancer metal-drug [11].
In continuation of radionuclidic homolog development of 
anti-proliferative metal complexes [12, 13], we were interested in 
developing a lanthanide-based labelled compound from an LaPQ3 
lead compound. Among radioactive lanthanides used in nuclear 
medicine, lutetium-177 is an interesting candidate for therapeutic 
protocols.
Owing to the suitable decay characteristics of lutetium-177 
[T1/2 = 6.73 d, Ebmax = 497 keV, Eg = 113 keV (6.4%), 208 keV 
(11%)] as well as the feasibility of large-scale production in ad-
equate specific activity and radionuclidic purity using a moderate 
flux reactor, 177Lu could be considered as a promising radionuclide 
in the development of Lu-PQ3 complex as a possible therapeutic 
radiopharmaceutical (Figure 1).
177Lu-radiopharmaceuticals have been used in the therapy of 
various diseases and malignancies, such as somatostatin receptor 
positive tumour radiotherapy [14], radioimmunotherapy [15], bone 
palliation therapy [16], and radiosynovectomy [17, 18].
In this research, 177Lu-PQ3 complex was prepared and the 
effects of various production conditions were investigated on its la-
belling yield. 177Lu-PQ3 complex solution was injected intravenously 
into normal rats for biodistribution determination.
Hassan Yousefnia, Amir R. Jalilian, S. Zolghadri, 
A. Bahrami-Samani, S. Shirvani-Arani, M. Ghannadi-Maragheh
Radiopharmaceutical Research and Development Lab (RRDL), 
Nuclear Science and Technology Research Institute (NSTRI), Tehran, Iran
[Received 17 II 2011; Accepted 18 II 2011]
50
Nuclear Medicine Review 2010, Vol. 13, No. 2
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Review
Material and methods
177Lu was produced with a specific activity of approximately 
70–80 mCi/mg and radionuclidic purity of 99.98% by irradiation 
of natural Lu2O3 targeted at a thermal neutron flux of approxi-
mately 4 × 1013 n/cm2.s for 5 days at the Tehran Research Reac-
tor (TRR). Whatman No. 1 paper was obtained from Whatman 
(Maidstone, UK) for instant thin layer chromatography (ITLC). 
Radio-chromatography was performed by using a Bioscan 
AR-2000 radio TLC scanner instrument (Bioscan, Washington, 
DC, USA). A high purity germanium (HPGe) detector coupled 
with a Canberra™ (model GC1020-7500SL) multichannel ana-
lyser and a dose calibrator ISOMED 1010 (Dresden, Germany) 
were used for counting distributed activity in rat organs. All other 
chemical reagents were purchased from Merck (Darmstadt, 
Germany). Calculations were based on the 208 keV peak for 
177Lu. All values were expressed as mean ± standard devia-
tion (Mean ± SD) and the data were compared using student 
T-test. Statistical significance was defined as P < 0.05. Animal 
studies were performed in accordance with the United Kingdom 
Biological Council's Guidelines on the Use of Living Animals in 
Scientific Investigations, 2nd ed. Male healthy rats were purchased 
from the Pasteur Institute, Tehran, Iran.
Production and quality control of 177LuCl3 solution
Lutetium-177 was produced by neutron irradiation of 1 mg 
of natural Lu2(NO3)3 (99.999% from Aldrich Co., UK) according 
to reported procedures [19] in the Tehran Research Reactor at 
a thermal neutron flux of 4 × 1013 n/cm2.s for 5 days. The specific 
activity of the produced 177Lu was 75 mCi/mg after 5d of irradia-
tion. The irradiated target was dissolved in 200 μl of 1.0 M HCl, to 
prepare 177LuCl3, and diluted to the appropriate volume with ultra 
pure water, to produce a stock solution of final volume of 5 ml. The 
mixture was filtered through a 0.22 μm biological filter and sent 
for use in the radiolabelling step. The radionuclidic purity of the 
solution was tested for the presence of other radionuclides using 
beta spectroscopy as well as HPGe spectroscopy for the detection 
of various interfering beta and gamma emitting radionuclides. The 
radiochemical purity of the 177LuCl3 was checked using 2 solvent 
systems for ITLC [A: 10mM DTPA pH.5 and B: ammonium acetate 
10%:methanol (1:1)].
Synthesis of 177Lu-PQ3 complex
The acidic solution (0.2 ml) of 177LuCl3 (111 MBq, 3 mCi) 
was transferred to a 5 ml-borosilicate vial and heated to dryness us-
ing a flow of N2 gas at 50–60°C. Fifty microlitres of PQ in absolute 
ethanol (1 mg/ml ª 274 nmoles) was added to the activity-con-
taining vial, and the mixture was diluted by the addition of normal 
saline (4.5 ml) followed by vortexing at 25°C for 30–60 min. The 
active solution was checked for radiochemical purity by ITLC. The 
final solution was then passed through a 0.22 mm filter and pH 
was adjusted to 5.5–7. 
Stability of 177Lu-PQ3 in final formulation
The stability of 177Lu-PQ3 in final preparation was determined 
by storing the final solution at 25°C for 2 days and performing 
frequent ITLC analysis to determine radiochemical purity. 
Stability of 177Lu-PQ3 in the presence of human 
serum
 The final 177Lu-PQ solution (200 μCi, 50 μl) was incubated in 
the presence of freshly prepared human serum (300 μl) and kept 
at 37°C for 2 days. The complex stability was assessed by size 
exclusion chromatography on a Sepharose column (1 × 30 cm). 
The column was equilibrated with PBS and eluted at a flow rate 
of 0.5 ml/min at room temperature; 1 ml fractions were collected 
and their activities were determined in a dose calibrator; also, the 
presence of serum proteins in each fraction was determined by 
Folin-Colciteau® method. The control samples were applied to the 
column separately, including Lu3+ cation, 177Lu-PQ complex, and 
human serum sample for retention time determination.
Quality control
For measuring radiochemical purity and radiolabelling yield, 
a 1 μl sample of the 177Lu-PQ3 complex was spotted on a chroma-
tography paper (Whatman No. 1) and developed in 10 mM DTPA 
solution (pH = 5) as the mobile phase. 
Biodistribution studies
The biodistribution of free Lu3+ cation as well as 177Lu-PQ3 
were determined in wild-type rats. For each piece, 100 μl (150 
μCi) of radioactive solution was injected directly into a normal rat 
through their caudal vein. The animals (n = 3) were sacrificed by 
CO2 asphyxiation at selected times after injection (2 to 48 h) and 
the percentage of injected dose in the tissues (brain, heart, liver, 
kidney, testis, spleen, lung, stomach, bladder, etc.) was determined 
by g-ray scintillation.
Results and discussion
Production and quality control of 177Lu
The radionuclide was prepared in a research reactor according 
to regular methods, with a range of specific activity 2.5–3 GBq/mg 
Figure 1. Possible chemical formula for 177Lu-PQ3.
51www.nmr.viamedica.pl
Hassan Yousefnia et al. Development of 177Lu-PQ3
Review
for radiolabeling use. After counting the samples on an HPGe 
detector for 5 hours, two major photons (6.4% of 0.112 MeV and 
11% of 0.208 MeV) were observed (Figure 2).
The radioisotope was dissolved in acidic media as a starting 
sample and was further diluted and evaporated to obtain the 
desired pH and volume followed by sterile filtering. 
The radiochemical purity of the 177Lu solution was checked in 
two solvent systems; in 10 mM DTPA, the free Lu3+ cation is com-
plexed to a more lipophilic LuDTPA form and migrates to a higher 
Rf, while small radioactive fraction remains at the origin, which could 
be related to other Lu ionic species not forming LuDTPA complex, 
such as LuCl4
–, etc. and/or colloids. 
On the other hand, ammonium acetate:methanol mixture 
was also used for the determination of radiochemical purity. The 
fast eluting species was possibly the ionic Lu-177 cations other 
than Lu3+, and the remaining fraction at Rf.0 was a possible 
mixture of Lu3+ and/or colloids. Due to the existence of 1% 
impurity in both cases, the existence of colloids is unlikely 
(Figure 3).
Preparation of 177Lu-PQ complex
The effect of various factors on the labelling yield of 177Lu-PQ 
was studied. In higher concentration no significant difference 
exists on labelling yield for added 177Lu-chloride activity (3 mCi). 
To investigate the effect of PQ concentration on labelling yield 
various amounts of the ligand were added to a fixed amount 
of activity. Labelling yield increased with increasing PQ amount 
(0.01–0.05 mg) and reached more than 99% when the ligand 
reached 0.05 mg after 30 minutes. ITLC showed that the complex 
is majorly prepared in 30 minutes with 99% radiochemical purity; 
the remaining 1% is possibly attributed to other Lu ionic spe-
cies which cannot react with PQ (Figure 4).
Stability of 177Lu-PQ complex in final product
The stability of the prepared 177Lu-PQ complex was checked 
up to 48 hours after preparation. The complex was stable in acidic 
media (pH = 5.5–7) and its radiochemical purity was above 99% 
even 48 hours after preparation. 
Stability of 177Lu-PQ complex in presence of human 
serum
A stability test was developed for the complex in the presence 
of human serum at 37°C. The mixture was then passed through 
a Sephadex column followed by elution with PBS, and each frac-
tion was checked for radioactivity and the presence of serum 
proteins by colorimetric method.
Biodistribution studies for 177Lu cation in rats
The animals were sacrificed by CO2 asphyxiation at selected 
times after injection (2, 4, 24, and 48 h). Dissection began by 
drawing blood from the aorta followed by removing heart, spleen, 
muscle, brain, bone, kidneys, liver, intestine, stomach, lungs, and 
skin samples. The tissue uptakes were calculated as the percent-
age of the area under the curve of the related photo peak per gram 
of tissue (% ID/g) (Figure 5).
The biodistribution of 177Lu cation was determined in 
wild-type animals for better comparison of 2–48 h post injec-
tion. The liver uptake of the cation is comparable to many other 
Figure 2. HPGe spectrum for Lu-177 chloride solution used in this study.
52
Nuclear Medicine Review 2010, Vol. 13, No. 2
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Review
radio-metals mimicking ferric cation accumulation; about 3 % 
of the cation accumulates in the liver after 48 h. The transferrin 
metal uptake and final liver delivery looks to be the possible 
route of accumulation. 
The blood content is low at all time intervals, and this shows the 
rapid removal of activity in the circulation. Lung, muscle, and skin 
do not demonstrate significant uptake while it is in accordance 
with other cation accumulation. A 4% bone uptake is observed 
for the cation, which remains almost constant after 48 h up to one 
week (data not shown). 
The spleen also has significant uptake, possibly related 
to reticuloendothelial uptake. As water-soluble cation, Lu3+, 
kidney plays an important role in excretion via urine, especially 
after 24 h.
Biodistribution studies for 177Lu-PQ3 in rats
The distribution of injected dose in rat organs up to 48 h after 
injection of 177Lu-PQ3 (60 μCi/100ul) solution was determined. 
Based on these results, it was concluded that the largest portion of 
injected activity of 177Lu-PQ3 was extracted from blood circulation.
The complex is majorly accumulated in the reticuloendothelial 
system, while small amounts of activity in blood, kidney, and bone 
demonstrate the absence of any free cation released from the 
complex and/or produced as secondary metabolite.
Figure 3. ITLC chromatograms of 177LuCl3 solution in DTPA solution (pH = 5) (up) and 10% ammonium acetate:methanol (1:1) solution (down) 
using Whatman no. 2.
Figure 4. ITLC chromatograms of 177LuCl3 (left) and 177Lu-PQ solution (right) on Whatman no. 2 paper using a methanol: water: acetic acid 
(4:4:2) mixture.
53www.nmr.viamedica.pl
Hassan Yousefnia et al. Development of 177Lu-PQ3
Review
Conclusions
The 177Lu-PQ complex was prepared with high radiochemi-
cal yield (> 99 %) at optimized conditions; 0.05 mg of PQ in the 
presence of 3 mCi Lu3+ chloride for 30–60 minutes. The prepared 
complex was stable in the final solution at room temperature and 
the presence of human serum at 37°C and can be used even 
24 hours after preparation. IV injection of 177Lu-PQ complex to 
male wild-type rats demonstrated activity distribution among rat 
tissues using sacrification showed different accumulation from 
free Lu cation; most of the 177Lu-PQ3 was accumulated in the re-
ticuloendothelial system. Liver and spleen are major dose-limiting 
tissues. Further experiments on the accumulation of 177Lu-PQ3 in 
tumour-bearing animals are underway.
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Figure 5. Percentage of injected dose per gram (ID/g %) of 177LuCl3 in wild-type rat tissues at 2, 4, 24, and 48 h post injection.
Figure 6. Percentage of injected dose per gram (ID/g %) of 177LuPQ3 in wild-type rat tissues at 4, 24, and 48 h post injection.
54
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Review
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Preparation and quality control of 177Lu-[tris(1,10-phenanthroline) lutetium(III)] complex for therapy

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Preparation and quality control of 177Lu-[tris(1,10-phenanthroline) lutetium(III)] complex for therapy

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