New Potential In Situ Anticancer Agent Derived from [188Re]rhenium Nitro-Imidazole Ligand Loaded 5th Generation Poly-L-Lysine Dendrimer for Treatment of Transplanted Human Liver Carcinoma in Nude Mice

Abstract

Hepatocellular carcinoma (HCC) is the most common primary liver tumor and one of the fifth most common
tumors worldwide. In this article we report the results of in vivo studies of potential anticancer agent "[188Re]
rhenium-ImDendrim" derived from [188Re]rhenium-nitro-imidazole-methyl -1,2,3-triazol-methyl-di-(2-pycolyl)amine as radioactive ligand loaded 5th generation poly-L-lysine denrimer (172,3 kDa, 20 nM).
Methods: 5.0 × 10⁶ cells were subcutaneously injected into mice. Once tumor established, 4 mice lots were
treated with a single dose of the test item (37, 74, 92.5 and 111 MBq of [188Re]rhenium-ImDendrim, respectively) compared to control lots (free [188Re]rhenium and non-radioactive ImDendrim). By the end of the study in six weeks post-test compound administration, the tumors were collected for histological analysis.
Results: The treatment was well tolerated. In fact, [188Re]rhenium-ImDendrim shows high significant anti-tumor property in this experimental cancer model even with the lowest dose of 37 MBq compared to control groups. These results were further confirmed by histological analysis. Large tumor mass only observed in tumor's sections from mice in the control groups, were disappeared in favour of collagen tissue in treated groups. In conclusion, this novel potential radiopharmaceutical agent has giving promising experimental results by showing an anti-tumoral activity in this experimental of liver cancer model in mice under the tested conditions.

Full text

OMICS International
Research Article
Drug Designing: Open Access
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ISSN: 2169-0138
Yang et al., Drug Des 2017, 6:1
DOI: 10.4172/2169-0138.1000144
Volume 6 • Issue 1 • 1000144
Drug Des, an open access journal
ISSN: 2169-0138
Keywords: Hepatocellular carcinoma (HCC); Dendrimer; In situ
anticancer agent; Nanomedicine
Introduction
Cancers are among the leading causes of morbidity and mortality
worldwide, with approximately 14 million new cases and 8.2 million
cancer related deaths in 2012 [1].
Hepatocellular carcinoma (HCC) is the most common primary
liver tumor and one of the h most common cancers worldwide
representing 7.9% of all malignancies [2]. In northern Europe the
incidence is less than 5 per 100,000 people. In areas of high incidence
such as China and South East Asia HCC is reported to be about 20 per
100,000 people [3].
Until now, the curative resection is the most eective therapy for
HCC. However, the percent of patients able to be resected is reported
to be between 10 and 50% due to impaired liver function and delayed
diagnosis [4-6]. Furthermore, recurrence rates remain high following
tumour resection with a dismal 8.9% ve-year recurrence-free
survival [7]. General chemotherapy and radiotherapy oer somewhat
unsatisfactory responsiveness. Consequently, there is a clinical need for
advances in loco-regional liver treatments in patients with HCC.
Currently, local therapies for HCC include transcatheter
hepatic arterial infusion chemotherapy (TACE transarterial
chemoembolization) and radiofrequency ablation; both treatments
with the intent of providing local control while preserving essential
vascular structures, liver parenchyma and adjacent organs [8-15]:
In the one hand, TACE (Transarterial Chemoembolization) is the
standard of care for patients with intermediate HCC as described by
the Barcelona Clinic Liver Cancer group. TACE involves the injection
of a chemotherapeutic agent and then a further agent that embolizes
the artery feeding the tumor thus attempting tumor necrosis via
*Corresponding author: Had Belhadj-Tahar, Nanomedicine Research and Expetise
Group, French Association for Medical Research Advancement, (AFPREMED-
Canceropole), 9 rue du professeur Antoine BAISSET, 31100 Toulouse, France, Tel:
33646772224; E-mail: belhadj-tahar@afpremed.org
Received February 05, 2017; Accepted February 21, 2017; Published February
27, 2017
Citation: Yang G, Sadeg N, Belhadj-Tahar H (2017) New Potential In Situ An-
ticancer Agent Derived from [188Re]rhenium Nitro-Imidazole Ligand Loaded 5th
Generation Poly-L-Lysine Dendrimer for Treatment of Transplanted Human Liver
Carcinoma in Nude Mice. Drug Des 6: 144. doi: 10.4172/2169-0138.1000144
Copyright: © 2017 Yang G, et al. This is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and
source are credited.
New Potential
In Situ
Anticancer Agent Derived from [188Re]rhenium
Nitro-Imidazole Ligand Loaded 5th Generation Poly-L-Lysine Dendrimer
for Treatment of Transplanted Human Liver Carcinoma in Nude Mice
Guanghua Yang1,2, Nouredine Sadeg1 and Had Belhadj-Tahar1*
1Nanomedicine Research and Expertise Group, French Association for Medical Research Advancement, (AFPREMED-Canceropole), France
2Research Center for Translational Medicine, East Hospital, School of Medicine, Tongji University, Shanghai 200120, China
Abstract
Hepatocellular carcinoma (HCC) is the most common primary liver tumor and one of the fth most common
tumors worldwide. In this article we report the results of in vivo studies of potential anticancer agent "[188Re]
rhenium-ImDendrim" derived from [188Re]rhenium-nitro-imidazole-methyl -1,2,3-triazol-methyl-di-(2-pycolyl)amine
as radioactive ligand loaded 5th generation poly-L-lysine denrimer (172,3 kDa, 20 nM).
Methods: 5.0 × 10⁶ cells were subcutaneously injected into mice. Once tumor established, 4 mice lots were
treated with a single dose of the test item (37, 74, 92.5 and 111 MBq of [188Re]rhenium-ImDendrim, respectively)
compared to control lots (free [188Re]rhenium and non-radioactive ImDendrim). By the end of the study in six weeks
post-test compound administration, the tumors were collected for histological analysis.
Results: The treatment was well tolerated. In fact, [188Re]rhenium-ImDendrim shows high signicant anti-tumor
property in this experimental cancer model even with the lowest dose of 37 MBq compared to control groups. These
results were further conrmed by histological analysis. Large tumor mass only observed in tumor's sections from
mice in the control groups, were disappeared in favour of collagen tissue in treated groups. In conclusion, this novel
potential radiopharmaceutical agent has giving promising experimental results by showing an anti-tumoral activity in
this experimental of liver cancer model in mice under the tested conditions.
reduction in blood supply and a cytotoxic eect. TACE, although a
palliative treatment option, has been shown to oer improved survival
in patients with unresectable HCC when compared to supportive
treatment alone [16].
In the other hand, Radiofrequency/microwave ablation is a potential
treatment option for HCC, especially in patients who are not t enough
to tolerate resection or transplant. It involves passing a needle directly
into the tumor through the skin, or directly at open or laparoscopic
surgery. Radiofrequency or microwave energy is administered to
heat the tumor and kill tumor cells. It is not a curative procedure but
provides improvement in long term outcomes in patients who would
not be able to tolerate curative procedures [2].
Otherwise the radioembolization is a local transarterial approach
that capitalizes on tumor hypervascularity to deliver high doses of
radiation therapy while preserving normal parenchyma [17-20].
is treatment has been used in HCC, colorectal cancers (CRC),
neuroendocrine tumors (NE) and a variety of other primary cancers
[21-29].

Citation: Yang G, Sadeg N, Belhadj-Tahar H (2017) New Potential In Situ Anticancer Agent Derived from [188Re]rhenium Nitro-Imidazole Ligand
Loaded 5th Generation Poly-L-Lysine Dendrimer for Treatment of Transplanted Human Liver Carcinoma in Nude Mice. Drug Des 6: 144.
doi: 10.4172/2169-0138.1000144
Page 2 of 7
Volume 6 • Issue 1 • 1000144
Drug Des, an open access journal
ISSN: 2169-0138
e radioembolization device consists of 20-40 µ particles (beads),
where the 90Ytrium is integrated in a glass or resin matrix. [90Y]Yttrium
is beta emitter with a physical half-life of 64 hours and means tissue
penetration is about of 2.5 mm [29,30].
e micron-sized beads loaded with [90Y] Yttrium (20-40 µM) are
placed in contact with the tumor but there are not diusible within the
tumor. ereby, in the presence of a large tumor volume, deep areas of
the tumor remain inaccessible for the treatment.
An alternative method involves with lipiodol as vehicle of β emitters
such as [131I]Iodine or [188Re]rhenium and could be promptly absorbed
by hyper vascularized tumor aer injection [31,32].
Recently, direct intratumoral injection of nonremovable radioactive
material has been proposed for treatment of hepatic tumors such
sulde [188Re]rhenium colloidal or dendrimers loaded [188Re]rhenium
complexes [33-35].
In this context, we have developed a potential in situ anticancer
treatment of hepatic tumors using [188Re]rhenium-nitro-imidazole derived
ligand loaded 5th generation poly-L-lysine dendrimer [35]. is method
has at least three advantages as a powerful therapeutic tool:
a) A spherical polycationic supramolecule dendrimer vector
whose diameter is adaptable to embolize neo-vascularization
of tumor stroma. In addition, the dendrimer injected directly
into or near the tumor, remains in the site of injection and then
could be considered as nano delivery system [36,37];
b) [188Re]Rhenium, an isotope with a 16.9 h half-life, has a
maximum beta energy compared to that of [90Y]Yttrium and
gamma emission of 155-KeV (15%) photon that is suitable for
imaging [38]. is β emitter complexed with a nitro-imidazole
ligand vector that is preferentially taken up by the hypoxic cells,
resulting in the radiotoxic eect optimization [39];
c) Providing new targeting anticancer agents in situ for treating
primary and/or metastatic tumors [35].
In this paper we report the preparation and the results of in vivo
preclinical studies of new potential in situ therapeutic agent “[188Re]
rhenium-ImDendrim” derived from [188Re]rhenium-nitro-imidazole-
methyl-1,2,3-triazol-methyl-di-(2-pycolyl)amine as radioactive ligand
loaded 5th generation poly-L-lysine denrimer (172,3kDa, 20nM).
Methods
e experiment agent “ImDendrim” used in this study is consisting
of 5th generation poly-L-lysine dendrimer (from Colcom, France)
mixed with nitro-imidazole-methyl-1,2,3-triazol-methyl-di-(2-pycolyl)
amine (Figure 1). All chemicals used in the synthesis of nitro-imidazole-
NH
NH
2
O
H
2
N
O
NH
2
H
2
N
N
N
N
NN
N
O
2
N
M
+
N
N
CO
CO
N
N
N
N
N
N
O
2
N
M
+
N
N
CO CO
N
NN
N
N
N
O
2
N
M
+
N
N
CO
NNNN
N
N
O
2
N
M
+
N
N
CO
CO
NNNN
N
N
O
2
N
M
+
N
N
CO
CO
N
N
N
N
N
N
O
2
N
Re
+
NN
CO
CO
CO
NNNN
N
N
O
2
N
M
+
N
N
CO
NNNN
N
N
O
2
N
M
+
N
N
CO
CO
NNNN
N
N
O
2
N
M
+
N
N
CO
CO
N
N
N
NN
N
O
2
N
Re
+
N
N
CO
CO
N
N
N
NN
N
O
2
N
M
+
N
N
CO
CO
M
+
CO
C
O
N
N
O
2
N
NN
N
N
N
N
HN
O
HN
HN
HN
O
NH
NH
2
O
NH
2
O
H
2
N
NH
2
H
2
N
O
NH
ONH
HN
O
NH
NH
2
NH
O
H
2
N
NH
2
O
NH
2
O
H
2
N
NH
NH
O
NH
O
NH
HN
O
HN
NH
2
O
NH
2
NH
2
O
NH
2
NH
2
NH
2
O
NH
2
NH
NH
2
O
NH
NH
2
O
H
2
N
NH
2
O
H
2
N
Dendrimer
Core
NH
ONH
HN
O
NH
NH
2
NH
O
H
2
N
NH
2
O
NH
2
O
H
2
N
NH
NH
2
O
NH
NH
2
O
H
2
N
NH
2
O
H
2
N
[188Re]Nitro-Im idazole Li gand
Figure 1: Structure of [188Re]rhenium-ImDendrim.
[188Re]rhenium-ImDendrim is composed by [188Re]rhenium nitro-imidazole-methyl-1,2,3-triazol-methyl-di-(2-pycolyl)amine as radioactive nitro-imidazole derived ligand
loaded 5th poly-L-lysine Dendrimer

Citation: Yang G, Sadeg N, Belhadj-Tahar H (2017) New Potential In Situ Anticancer Agent Derived from [188Re]rhenium Nitro-Imidazole Ligand
Loaded 5th Generation Poly-L-Lysine Dendrimer for Treatment of Transplanted Human Liver Carcinoma in Nude Mice. Drug Des 6: 144.
doi: 10.4172/2169-0138.1000144
Page 3 of 7
Volume 6 • Issue 1 • 1000144
Drug Des, an open access journal
ISSN: 2169-0138
methyl-1,2,3-triazol-methyl-di-(2-pycolyl)amine were purchased from
Sigma-Aldrich, France. [188Re]rhenium (188ReO4 Na) was eluted from
an 188W/188Re Generator (Institute for Radioelements, Belgium).
Synthesis of nitro-imidazole-methyl -1,2,3-triazol-methyl-di-
(2-pycolyl)amine (nitroimidazole derived ligand)
e synthesis of ligand procedure is summarized in Figure 2. e
chloro-alkyl-nitro-imidazole compound (2) was obtained by adding the
amine function on the chloro methyl imidazole (1). e azido-methyl-
nitroimidazol compound (3) was obtained by substitution of the
chlorine atom by an azide group. e nal compound nitro-imidazole-
methyl -1,2,3-triazol-methyl-di-(2-pycolyl)amine (5) was obtained by
dissolving propargyl di-(2-picolyl)amine (4) with the azido-methyl-
nitruroimidazol compound (3) in a solvent mixture dioxane/water at
100°C in the presence of copper sulfate and sodium ascorbate.
Protocol
Paraformaldehyde (14.6 g, 0.49 mol) was added to chloro methyl
imidazole (50 g, 0.44 mol) in 500 ml of chloroform and the reaction
was kept under stirring for 48 hours at room temperature. en, thionyl
chloride (57.9 g, 0.49 mol) in dry dichloromethane was added drop wise
under an atmosphere of argon. e white precipitate of azido-methyl-
nitro-imidazole was ltered, washed with dichloromethane and dried.
Propargyl di-(2-picolyl)amine (8.5 g, 36 mmol) was added to azido-
methyl-nitroimidazol (6 g, 36 mmol) in a solvent mixture dioxane/water
at 100°C in the presence of copper sulfate(1.7 g, 6.8 mol) and sodium
ascorbate (2.9 g, 15 mmol) and the reaction was kept for 4 h at 90°C. e
suspension was ltered and dried. en obtained nitro-imidazole-methyl-
1,2,3-triazol-methyl-di-(2-pycolyl)amine compound was redissolved in
450 ml of tetrahydromethylfuran and the organic phase was washed by 350
ml of ammonium solution buered at pH12. Aer tetrahydromethylfuran
solvent removal, the resulted brown oil was dried at high vacuum (8 g,
yield≈56%). 1H NMR (D2O, 200 MHz, δ/ppm relative to TMS): H2: 8.3 ±
0.2 ppm (d), Ha,a’: 8.23 ± 0.2 ppm (d), H3: 7.99 ± 0.2 ppm (s), H1: 7.97 ± 0.2
ppm (d), Hc,c’: 7.62 ± 0.2 ppm (t); Hd,d’: 7.39 ± 0.2 ppm (d); Hb,b’: 7.11, ± 0.2
ppm (t), NCH2N: 6.57 ± 0.2 ppm (s).
Preparation of nitro imidazole derived ligand/dendrimer
samples for freeze-drying
50 mg of polylysine dendrimer were dissolved in 4 ml of sterile
saline purged with N2 gas. 2 ml of ethanol solution of nitro-imidazole
derived ligand (15 mg/ml) was drop wise added under vigorous stirring
to obtain a homogenous solution. e volume was completed to 10 ml
using N2 purged saline. e solution was dispensed in 1ml quantities
into sterile penicillin vials and tted with sterile rubber closures. e
vials were transferred to the freeze-dryer and the process continues for
24 h. e vials were closed under dry sterile nitrogen gas and stored at
6–8°C.
Labelling with [188Re]Rhenium
In a typical radiolabelling procedure, 350 µL of perrhenate saline
solution (1,850 MBq of [188Re] ReO4, NaCl), 100 µL of hydrochloric
acid at 2 N and 250 µL of 2-(N-morpholino)-ethane-sulfonic acid
hydrate (MES) at 0.5 N were added into the vial containing borane
ammonia complex (3.5 mg) and the mixture was purged with CO gas
and heated at 60°C for 20 min. e lyophilized nitro-imidazole derived
ligand/dendrimer already dissolved in 500 µl of saline were added to
the previous vial and then heated at 60°C for 1 h. e radiochemical
purity of Imdendrim was controlled by TLC coupled with Gamma
radioactivity scanner (Raytest, Germany).
In Vi vo Tests
e in vivo experimental protocols were carried out in accordance
with the strict French ethical requirements relating to animal testing.
Cell culture and in viv o graing
e human HCC tumor cell line, HepG2 (obtained from ATCC),
was routinely cultured at 37°C, 5% CO2, containing 10% fetal calf
serum (FCS), 1% penicillin/streptomycin, 1% sodium pyruvate (Sigma-
Aldrich). e tumor cells required passaging to ensure their viability
and reach the required amount of cells. e log-growing tumor cells
were trypsinized, counted and used for these studies. en 5.0 × 106
cells/200 µL were subcutaneously injected into mice.
Subject/experimental animals
60 Athymic nude male mice aged 4-6 weeks (from Harlan
Laboratories, France) were used for this study. e animals were housed
in a climate-controlled room with a 12/12 h light cycle. e subjects had
free access to food and water during housing.
NNH
O2N
O2N
N
N
N
N
N
N
N
N
Cl
O2N
N
NNaN3
DMF, T°C
3
NN
N
CuSO4, NaAsc.,
Dioxane/H2O
4
H2CO
SOCl2, CHCl3
N3
O2N
N
N
N3
O2N
N
N
(a)
(b)
(c)
(d)
(a')
(b'
)
(c')
(d')
(1)
(2)
(3)
(5)
(6)
(7) (8)
(8')
123
5
Figure 2: Synthesis of nitro-imidazole derived Ligand*.
Where: *Nitroimidazole derived Ligand : Nitro-imidazole-methyl -1,2,3-triazol-methyl-di-(2-pycolyl)amine

Citation: Yang G, Sadeg N, Belhadj-Tahar H (2017) New Potential In Situ Anticancer Agent Derived from [188Re]rhenium Nitro-Imidazole Ligand
Loaded 5th Generation Poly-L-Lysine Dendrimer for Treatment of Transplanted Human Liver Carcinoma in Nude Mice. Drug Des 6: 144.
doi: 10.4172/2169-0138.1000144
Page 4 of 7
Volume 6 • Issue 1 • 1000144
Drug Des, an open access journal
ISSN: 2169-0138
Tumor inhibiting study
Sixty nude mice bearing HepG2 xenogra tumors were divided
into six groups. Four groups were treated with a 0.1 ml of [188Re]
rhenium-ImDendrim at doses of 37, 74, 92.5 and 111 MBq by a single
intratumoral injection. Two groups served as the controls and were
injected with free [188Re]rhenium and none radioactive ImDendrim
respectively. e tumors were measured every 2 or 3 days with vernier
calipers in two dimensions.
e mean tumor volumes (MTV and MTV+SEM) will be by using
the following formula:
Tumor volume=(Width2. Length)/2
In vivo SPECT/CT imaging
Aer 3 and 24 h post in situ administration of [188Re]rhenium-
ImDendrim, 3 mice from each group were anesthetized using 80 mg/
kg body weight for Ketamine and 8 mg/kg body weight for Xylazine
injected intraperitoneally and successively placed in hybrid SPECT/CT
scanner for in vivo imaging (eXplore speCZT, GE Healthcare, USA).
Anatomical CT imaging was performed initially followed by SPECT
imaging. us to being reconstructed into a single image, the CT data
was used for attenuation correction of SPECT images. SPECT/CT image
analysis was performed using MicroView soware (GE Healthcare Inc.)
[40].
Clinical Evaluation
Daily clinical examination of all animals was carried out including:
behaviour, signs of suering (hunching, convulsions, weakening,
diculty for moving or feeding, etc.). Determination of body weight
twice a week for each mouse, a body weight curve was designed (Mean
± SD).
Histological Study
At the end of experiment (six weeks post injection), all animals
were anesthetized and then sacriced according to ethical guidelines
in France. en, the tumors were immediately collected and six
representative tumor sections of each group were xed in 4% formalin
and paran-embedded for histological analysis.
Statistics
Data were expressed as means ± standard deviation (SD). Statistical
signicance of mean values in two groups or treatment eects were
analyzed with Student’s t-test or one-way ANOVA using Microso
Excel program. A p-value less than 0.05 are considered as signicant.
Results
e experiment [188Re]rhenium-ImDendrim ([188Re]rhenium-
nitro-imidazole-methyl-1,2,3-triazol-methyl-di-(2-pycolyl)amine
loaded poly-L-lysine dendrimer) injected in situ into mice has a high
radiochemical purity yield estimated at 99.5%.
Under this experimental condition, no clinical signs and no
mortality were observed in any treated groups.
Mean body weight was the same in all groups at the injection of
treatment. Weight loss was more marked in treated groups as compared
to untreated groups. e dierence became signicant aer 15 days
and remained signicant thereaer for all the measurements shown in
Figure 3. Clinically, All treated mice remained in complete remission
and in apparent good health for 6weeks, until end of experiment.
Antitumor eect
At the time of the injection, tumor volumes were similar in the six
groups. As observed in Figure 4, all tumors in treated groups continued
to grow for rst week, reaching a maximum size of 235 mm3 ± 104 mm3.
By day 14, the dierences between the tumor volumes in the treated and
control groups were very signicant (p<0.002). In contrast, there is no
signicant dierence between the control Groups, the average volumes
of tumors injected with free [188Re]rhenium and non-radioactive
Figure 3: Mice body weight (BW) of HepG2 liver tumor bearing mice.
Where: Group 1 (GI): Non-radioactive ImDendrim; Group 2 (GII): 37 MBq of free [188Re]rhenium per mouse; Group 3 (GIII): 37 MBq of [188Re]rhenium-ImDendrim per
mouse; Group 4 (IV): 74 MBq of [188Re]rhenium-ImDendrim per mouse; Group 5 (GV): 92.5 MBq of [188Re]rhenium-ImDendrim per mouse; Group 6 (GVI): 111 MBq of
[188Re]rhenium-ImDendrim per mouse

Citation: Yang G, Sadeg N, Belhadj-Tahar H (2017) New Potential In Situ Anticancer Agent Derived from [188Re]rhenium Nitro-Imidazole Ligand
Loaded 5th Generation Poly-L-Lysine Dendrimer for Treatment of Transplanted Human Liver Carcinoma in Nude Mice. Drug Des 6: 144.
doi: 10.4172/2169-0138.1000144
Page 5 of 7
Volume 6 • Issue 1 • 1000144
Drug Des, an open access journal
ISSN: 2169-0138
ImDendrim were 1,600 ± 90 and 1,341 ± 473 mm3. In comparison,
the volumes injected with 37, 74, 92.5 and 111 MBq [188Re]rhenium
ImDendrim were 204 ± 55, 187 ± 74 and 178 ± 47 and 96 ± 40 mm3,
respectively. ese dierences are further illustrated in Figure 4. In all
mice, tumor remission was complete, with no evidence of relapse that
conrmed by histological analyses. e large tumor mass observed in
control group was replaced in treated mice by scar and collagen tissue
which is apparent in Figures 5a and 5b. ere is no signicant dierence
observed between the treated groups.
In vivo SPECT/CT imaging
24 h postinjection of [188Re]rhenium-ImDendrim, the quasi total
administered radioactivity was retained locally in the injection site. As
shown in Figure 6, time-course study showed no signicant diusion
outside the injection area and notably no signicant uptake in dierent
organs including lung, heart, liver kidney and brain.
Discussion
In situ injection of no removable radioactive material using
dendrimers is a promising alternative method for tumor treatment
because it can deliver high doses of radiation to target sites and prevent
normal tissues and no target organs from radiation while delivering
radiopharmaceutical to target sites [33-35].
In this context, the purpose of this investigation was to evaluate
the therapeutic eectiveness of [188Re]rhenium-ImDendrim consisting
of [188Re]rhenium nitro-imidazole-methyl-1,2,3-triazol-methyl-di-(2-
pycolyl)amine as radioactive ligand loaded 5th generation poly-L-lysine
2000
1800
1600
1400
1200
1000
800
600
400
200
0
0 10 20 30 40 50 60
Time (Days)
Volume of Tumor (mm
3
)
1
2
3
4
5
6
Figure 4: Tumor volume measurements of HepG2 liver tumor bearing mice.
Where: Group 1: Non-radioactive ImDendrim; Group 2: 37 MBq of free [188Re]rhenium per mouse; Group 3: 37 MBq of [188Re]rhenium-ImDendrim per mouse; Group 4: 74
MBq of [188Re]rhenium-ImDendrim per mouse; Group 5: 92.5 MBq of [188Re]rhenium-ImDendrim per mouse; Group 6: 111 MBq of [188Re]rhenium-ImDendrim per mouse.
AB
Figure 5: Comparative Histological analyses on tumors in control groups test (a) and treated groups (b) The large tumor mass observed in control group (5a) was
replaced in treated mice by scar and collagen tissue (5b).

Citation: Yang G, Sadeg N, Belhadj-Tahar H (2017) New Potential In Situ Anticancer Agent Derived from [188Re]rhenium Nitro-Imidazole Ligand
Loaded 5th Generation Poly-L-Lysine Dendrimer for Treatment of Transplanted Human Liver Carcinoma in Nude Mice. Drug Des 6: 144.
doi: 10.4172/2169-0138.1000144
Page 6 of 7
Volume 6 • Issue 1 • 1000144
Drug Des, an open access journal
ISSN: 2169-0138
dendrimer on mice bearing HCC HepG2 tumors. e charged cationic
poly-L-lysine dendrimer complex showed high retention in the site of
injection and no local or general toxicity aer single or iterative in situ
injection [41].
Our previous in vivo studies on radioactive 5th generation poly-L-
lysine dendrimer labelled with [188Re]rhenium or [99mTc] Technetium
injected intravenously at 100 mg/L in the rats, showed no acute or sub-
acute toxicity and the pulmonary uptake was transient with its constant
clearance l=0.57 h-1 (half-life=1.2 h) and important constant uptake in
liver (4.74% of injected dose/g) [42].
In the present experiment, [188Re]rhenium-ImDendrim was well
tolerated while it was intra tumor injected as single dose varying from
37 to 111 MBq on the mice bearing tumors.
Indeed, no clinical signs and no mortality were observed in any
treated groups. Concerning mice body weight evolution (Figure 3), all
treated groups by 2 weeks post injection of [188Re]rhenium-ImDendrim
had signicant higher body weight than the control groups in
connection with health improvement of the treated mice. ese results
suggest that the used doses under current experimental conditions did
not induce notable toxicity in mice.
According to the obtained results, in vivo SPECT/CT imaging
study, quasi total [188Re]rhenium-ImDendrim is retained in tumor
volume aer its in situ administration in mice and no signicant spread
toward other organs was observed (Figure 6). Such local retention of
radioactive [188Re]rhenium-Imdendrim could dramatically reduce
its side-eect. In addition, the [188Re]rhenium beta particles has the
average penetration in tissue of 3.8 mm (maximum 11 mm) and results
in a more homogeneous tumor distribution of the radiation dose
[33,34].
[188Re]rhenium-ImDendrim showed high signicant anti-tumor
property in this experimental cancer model even with the lowest dose
of 37MBq of activity per mouse, in comparison of injection of non-
radioactive ImDendrim or free [188Re]rhenium which do not have any
anti-tumor eect. Without poly-L-lysine dendrimer as nano carrier, the
free complex of [188Re]rhenium or [99mTc] Technetium nitro-imidazole
derived ligand (Nitro-imidazole-methyl-1,2,3-triazol-methyl-di-
(2-pycolyl)amine) has a low tumor bioavailability due to its large
distribution volume estimated at 167 L.kg-1 (unpublished data). e
charged poly-L-lysine groups of dendrimer electro-statically attract the
[188Re]rhenium -nitro-imidazole derived ligand and limit its diusion
rate. e rate diusion seems to be under the inuence of the in situ
environment since the released [188Re]rhenium-nitro-imidazole derived
ligand could be specically attracted by the hypoxic tumoral cells [39].
In this condition, e tumor bioavailability of this [188Re]rhenium-
nitro-imidazole derived ligand mixed with poly-L-lysine dendrimer
could be enhanced in presence of hypoxic condition specially observed
in case of large tumor volume.
At 6t h week post treatment in all mice, tumor remission was
complete, with no evidence of relapse that conrmed by histological
analyses.
In conclusion, this novel potential therapeutic agent has giving
promising experimental results by showing an anti-tumor activity in this
experimental liver cancer model in mice under the tested conditions.
Further works concerning the optimization of injected radioactive
ImDendrim dose in terms of the tumor volume are in progress.
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Citation: Yang G, Sadeg N, Belhadj-Tahar H (2017) New Potential In Situ Anticancer Agent Derived from [188Re]rhenium Nitro-Imidazole Ligand
Loaded 5th Generation Poly-L-Lysine Dendrimer for Treatment of Transplanted Human Liver Carcinoma in Nude Mice. Drug Des 6: 144.
doi: 10.4172/2169-0138.1000144
Page 7 of 7
Volume 6 • Issue 1 • 1000144
Drug Des, an open access journal
ISSN: 2169-0138
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Citation: Yang G, Sadeg N, Belhadj-Tahar H (2017) New Potential In Situ An-
ticancer Agent Derived from [188Re]rhenium Nitro-Imidazole Ligand Loaded 5th
Generation Poly-L-Lysine Dendrimer for Treatment of Transplanted Human Liv-
er Carcinoma in Nude Mice. Drug Des 6: 144. doi: 10.4172/2169-0138.1000144