Therapeutic efficacy of 188Re-MN-16ET lipiodol in an animal
model of hepatocellular carcinoma
Ping-Wun Huang
•
Shih-Chung Tsai
•
Tsai-Yueh Luo
•
Chia-Hung Kao
Introduction
In recent years, rhenium-188 (Re-188) has emerged as an
extremely versatile therapeutic radionuclide because it has
several advantages including low cost, production via
generator, and additional low gamma energy. Various
Re-188-based radiopharmaceuticals were developed to
treat inoperable liver malignancy [1–5]. Among these
Re-188-based radiopharmaceuticals for treatment of liver
tumors, Re-188 HDD lipiodol was the first to be introduced
in clinical studies by an International Atomic Energy
Agency (IAEA)-sponsored group of hospitals located in 8
different countries in Asia and South America. The results
of phase I and II studies were very encouraging and suggest
Re-188 HDD lipiodol is a safe and cost-effective method to
treat primary hepatocellular carcinoma (HCC) via the
transarterial route [5–8]. However, one research group
reported that acetylated HDD provided better kit stability
for hepatoma therapy than non-acetylated HDD [9].
In our recent study, we labeled Re-188 with a new N2S2
tetradentate ligand, N-[2-(triphenylmethyl)thioethyl]-3-aza19-ethyloxycarbonyl-3-[2-(triphenylmethyl)thioethyl]octadecanoate
(H3MN-16ET) and successfully developed a new
rhenium-based radiopharmaceutical (Re-188 MN-16ET
lipiodol) for the treatment of liver malignancy [10]. In that
study, the stability of Re-188 MN-16ET lipiodol was very
good. The biodistribution and micro-single-photon-emission
computed tomography/computed tomography image data
showed that Re-188 MN-16ET lipiodol via intra-hepatic
arterial injection was selectively retained at the tumor site. In
that article, we concluded that Re-188 MN-16ET lipiodol has
•
Wan-Yu Lin
the potential to be a therapeutic radiopharmaceutical for
hepatoma treatment.
In this study, we further evaluated the therapeutic efficacy
of this new radiopharmaceutical by measuring tumor
response and survival times in rats with liver tumors after
intra-hepatic arterial injection of Re-188 MN-16ET
lipiodol.
Materials and methods
Preparation of 188Re-MN-16ET/lipiodol
All laboratory chemicals were of reagent grade and
obtained from commercial sources. 2-Thiothylamine
hydrochloride and 16-bromohexadecanoic acid were purchased
from Sigma-Aldrich. Carrier-free 188Re was eluted
with normal saline from a 188W/188Re generator system
manufactured by the Institute of Nuclear Energy Research
(Taiwan).The detailed procedure for the synthesis of 188ReMN-16ET/lipiodol was described in our previous study
[10]. The formulation of 188Re-MN-16ET was carried out
using glucohepatonate as the transchelating agent and
stannous chloride as the reducing agent, then extracting
with lipiodol. The reaction was performed at 95 _C and
500 rpm with shaking for 1 h. The radiochemical purity
was evaluated by thin layer chromatography (TLC) with
silica-gel as the stationary phase and two kinds of developing
solution (ethyl acetate and normal saline). The
radiochemical purity of Re-188 MN-16ET lipiodol after
the extraction procedure was analyzed to be greater than
95 % and it remained stable for 24 h.
Animals and tumor cell line
Male rats (Sprague–Dawley rats) weighing 200–250 g
were fed on a standard chow diet and given water ad libitum.
An N1-S1 hepatoma cell line (ATCC, Maryland,
USA) was used for tumor implantation. This study was
approved by the Institutional Animal Care and Use Committee
of Taichung Veterans General Hospital. The tumor cells were routinely cultured in Dulbecco’s
modified eagle
medium (Gibco, Paisley, UK) mixed with 5 % fetal bovine
serum, 1 % L-glutamine, and 20 % horse serum. After
growing exponentially for 1 week, a concentration of
approximately 4 9 106 cells per ml was established. The
cell viability was more than 90 %, as determined by trypan
blue exclusion.
Inoculation
A sub-xyphoid laparotomy, 1.5–2 cm in length, was performed
to expose the left and right lobes of the rat liver.
Using a 27-gauge needle, a tumor cell suspension containing
4 9 107 cells in a volume of 0.1 ml was injected
slowly into one of the hepatic lobes under the liver capsule,
raising a visible pale wheal. The puncture site was gently
compressed for 15 s with cotton gauze to prevent bleeding.
Then, the wound was closed in layers. Two weeks after
inoculation, laparotomy was performed to check tumor
growth (Fig. 1). The tumors in sizes from 15 to 20 mm
were chosen for study.
Study of therapeutic efficacy
Twelve male rats bearing hepatic tumors were divided into
three groups (4 rats in each group) to evaluate the efficacy
of treatment. In Group 1, the rats were injected via hepatic
arteries with 18.5 MBq (0.5 mCi) Re-188 MN-16ET lipiodol
in a volume of 0.1 ml. In Group 2, the rats were
injected via hepatic arteries with 0.1 ml of lipiodol. In
Group 3, the rats were injected via hepatic arteries with
0.1 ml of normal saline. Tumor size was measured by liver sonography, Acuson 128 9 P computed
sonography (Philips),
before injection, and at 2, 4, and 8 weeks after
injection. The maximum length and width of the lesion
were measured by the same experienced ultrasound physician
using the same machine. Survival time was calculated
from the day of treatment to 60 days after treatment.
The response to treatment was classified according to the
survival time and change in tumor size from pre-treatment
to the 60th day after treatment as follows: (1) good
response: rats survived for more than 60 days and the
tumor size decreased in the 8th week after treatment when
compared with the baseline tumor size; (2) poor response:
any condition less than good response or survival time less
than 60 days. Fisher’s exact, two-tailed test was used to
calculate the differences in response rates.
Analyses were performed using the Statistical Package
for the Social Sciences (version 15.1; SPSS, Inc., Chicago,
IL, USA) and SAS version 9.1.3 software (SAS Institute,
Cary, NC, USA).
Results
Table 1 shows the changes of tumor size, therapeutic
response and survival time in the rats that were treated with
Re-188 MN-16ET lipiodol, lipiodol or normal saline
(control group). The tumor decreased in size after an intrahepatic
arterial injection of 18.5 MBq of Re-188 MN-16
ET lipiodol (Fig. 2, upper row). In contrast, the tumors
continuously grew in the rats which received 0.1 ml of
normal saline or 0.1 ml of lipiodol as treatment (Fig. 2,
lower row). All the rats showed good response to the
treatment with Re-188 MN-16ET lipiodol compared to the
rats in the normal saline and lipiodol groups, which all
showed poor response. There was a statistical difference in
the response rate between the Re-188 MN-16ET lipiodol
group and the normal saline group (or lipiodol group) with
a p value of 0.0142 by a Fisher’s exact, two-tailed test. No
statistical difference in the response rate was noted
between the normal saline group and the lipiodol group.
In the survival evaluation (Fig. 3; Table 2), all rats in
the normal saline group were dead before the end of the
experiment with the shortest survival time being 15 days
and the longest survival time 43 days (mean 28.2 days). Of
the four rats in the lipiodol group, three (75 %) were dead
before the end of the experiment with the shortest survival
time being 39 days and the longest survival time 60 days
(mean 47.7 days). The rats in the Re-188 MN-16 ET group,
in contrast, were all alive at the end of the experiment (60th
day after treatment). Using Kaplan–Meier survival curves
and the log-rank test, we found a significant difference in
the survival time between the Re-188 MN-16ET and the
normal saline group with a p value of 0.0067. There was
also a statistical difference in the survival time between the
Re-188 MN-16ET group and the lipiodol group with a
p value of 0.04. The rats treated with lipiodol also had
better survival than the rats treated with normal saline with
a p value of 0.034.
Discussion
Transhepatic arterial embolization (TAE) is a common
alternative treatment for an unresectable liver tumor.
Lipiodol, an ethyl ester of poppyseed oil fatty acids
(37–39 % by weight of iodine), has been used for many
years as embolitic material in TAE, and has been found to
be selectively retained in the foci of HCC [11]. Lipiodol labeled with various radioisotopes has been
applied in an
attempt to achieve better therapeutic effectiveness in HCC.
The most obvious choice in the matter naturally is iodine
131 and I-131 lipiodol indeed has proven to be a valuable
treatment option in the management of HCC [12, 13].
However, I-131 has some disadvantages, including its long
half-life (8.02 days) and emission of high energy gamma
rays (364 keV), which result in inappropriate irradiation to
surrounding tissue. Furthermore, the emission of lowenergy
beta rays (606 keV) resulting in low intratumoral
penetration is considered as another drawback. Yttrium-90
(Y-90) is considered to be a better radiotherapeutic candidate
than I-131. Y-90 has several advantages over I-131
including a shorter half-life (64 h), greater suitability for
therapy and a longer beta energy range (2.3 MeV) sufficient
to kill cells.
Re-188 has similar beta energy (2.2 MeV) characteristics
to Y-90, with a shorter physical half-life (17.4 h). In
addition, Re-188 emits 15 % 155 keV gamma rays and can
be produced by a W-188/Re-188 generator system. The
short half-life character of Re-188 can effectively reduce
the system irradiation secondary to escape from the treated
target. Sometimes, the escape of the radiopharmaceutical
from the treated target can result in severe adverse
events. CIS Bio International (Gif-sur-Yvette, France), manufacturer of I-131 lipiodol (Lipiocis), has
informed
health care professionals that the incidence of radiation
pneumonitis associated with the use of Lipiocis appears
notably higher (2 %) than initially observed in clinical
trials (0.5) [14]. These interstitial pneumopathies usually
manifest themselves approximately 1 month following
I-131 lipiodol injection and constitute a severe clinical
condition that generally leads to respiratory failure. In
contrast, the 15 % 155 keV gamma rays emitted from Re188 can be utilized to monitor the biodistribution of Re-188
lipiodol and to calculate radiation levels in various organs.
The use of a 188W/188Re generator system increases the
availability of Re-188 clinically. Therefore, we considered
Re-188 lipiodol to be an ideal radiotherapeutic drug for the
treatment of HCC.
In our study, the new tetradentate ligand H3MN-16ET
was proven to easily conjugate with the Re isotope and
showed good solubility in lipiodol, which was considered
to be owing to the following characteristics: (1) the N2S2
structure easily conjugates with Re-188, (2) the triphenylmethyl
protective group increases ligand stability, and (3)
the long alkyl esterified chain enhances the lipophilicity in
the lipiodol phase. According to our data, only the rats
treated with Re-188 MN-16ET lipiodol showed good tumor
response when compared with the rats in the lipiodol group
and the normal saline group. There were significant differences
in the response rates between the Re-188 MN16ET lipiodol group and the lipiodol group or the normal
saline group. In most rats treated with Re-188 MN-16ET
lipiodol, the response was obvious in the second week and
lasted to the eighth week post-treatment. Although poor
tumor response was noted in all rats in the lipiodol group
and normal saline group, the tumors apparently grew more
slowly in the lipiodol group than those in the normal saline
group. As a matter of fact, the rats treated with lipiodol
showed better survival time than the rats that received
normal saline as treatment with a p value of 0.034. The
results confirmed the effect of lipiodol alone in the treatment
of liver tumors although the effect was not satisfactory.
With the additional radiation effect from the Re-188,
the survival in the Re-188 MN-16ET lipiodol group was
significantly longer than that in the normal saline group
with a p value of 0.0067. In addition, the rats in the Re-188
MN-16ET lipiodol group also showed statistically better
survival than the rats in the lipiodol only group.
The therapeutic effect of radiation mainly depends on
the total radiation dose to the tumor. A small dose of a
radioisotope with a long half-life provides a high radiation
dose to the target. A large dose of a radioisotope with a
short half-life is usually needed to produce adequate radiation
therapy. In a recent study, McCann et al. [15] treated
unresectable liver tumors in 143 patients using an intraarterial
injection of Y-90 microspheres (half-life 2.7 days).
The mean treatment dose of Y-90 microspheres was
0.71 GBq (range 0.07–1.6 GBq). In our study, we intraarterially
injected 18.5 MBq of Re-188 MN-16ET lipiodol
into 200-g rats. With a 60-kg patient, we estimate the dose
would be 5.55 GBq. In our study, the high dose of Re-188
MN-16ET lipiodol had a good therapeutic effect and prolonged
the life of the rats although the physical half-life of
Re-188 is only 17.4 h. This property of Re-188 may be like
the proverbial ‘‘two-edged sword’’—it can both help and
hurt. The physical half-life of Re-188 may be short when
compared with the biological half-life of lipiodol in liver
tumors. However, the short physical half-life of Re-188
reduces the system irradiation secondary to escape from the
treated target and allows for higher doses compared to
doses with long-lived radionuclides.
Although Re-188 MN-16 ET lipiodol has potential for
use as a radiopharmaceutical for the treatment of liver
tumors, further evidence of its efficacy is needed before it
can be applied in routine clinical settings. The 188W/
188Re generator used in our study was produced by the
Institute of Nuclear Energy Research (Taiwan). This type
of generator can be purchased from the Isotopes Distribution
Office (IDO) at the Oak Ridge National Laboratory
(ORNL), Oak Ridge, TN, USA. However, these generator
systems have not yet been approved by either the U.S.
Food and Drug Administration (FDA) or the Taiwan FDA
as a commercial radiopharmaceutical for routine use.
Conclusion
In conclusion, our data suggest that this new radiopharmaceutical,
Re-188 MN-16ET lipiodol, has good response
and provides long survival time in rats with liver tumors
when compared with the control groups. We consider Re188 MN-16ET lipiodol has a very good potential to be a
therapeutic radiopharmaceutical for hepatoma treatment.