Characterization of a new class of androgen receptor antagonists with potential therapeutic application in advanced prostate cancer

Article abstract of DOI:10.1158/1535-7163.MCT-13-0422-T

The human androgen receptor plays a major role in the development and progression of prostate cancer and represents a well-established drug target. All clinically approved androgen receptor antagonists possess similar chemical structures and exhibit the s

Full text of DOI:10.1158/1535-7163.MCT-13-0422-T

Published OnlineFirst September 12, 2013; DOI: 10.1158/1535-7163.MCT-13-0422-T
Molecular
Cancer
Therapeutics
Small Molecule Therapeutics
177Lu-EC0800 Combined with the Antifolate Pemetrexed:
Preclinical Pilot Study of Folate Receptor Targeted
Radionuclide Tumor Therapy
1
1
2
1
€
Josefine Reber , Stephanie Haller , Christopher P. Leamon , and Cristina Muller
Abstract
Targeted radionuclide therapy has shown impressive results for the palliative treatment of several types of
cancer diseases. The folate receptor has been identified as specifically associated with a variety of frequent
tumor types. Therefore, it is an attractive target for the development of new radionuclide therapies using folate-
based radioconjugates. Previously, we found that pemetrexed (PMX) has a favorable effect in reducing
undesired renal uptake of radiofolates. Moreover, PMX also acts as a chemotherapeutic and radiosensitizing
agent on tumors. Thus, the aim of our study was to investigate the combined application of PMX and the
therapeutic radiofolate 177Lu-EC0800. Determination of the combination index (CI) revealed a synergistic
inhibitory effect of 177Lu-EC0800 and PMX on the viability of folate receptor–positive cervical (KB) and ovarian
(IGROV-1) cancer cells in vitro (CI < 0.8). In an in vivo study, tumor-bearing mice were treated with 177Lu-EC0800
(20 MBq) and a subtherapeutic (0.4 mg) or therapeutic amount (1.6 mg) of PMX. Application of 177Lu-EC0800
with PMXther resulted in a two- to four-fold enhanced tumor growth delay and a prolonged survival of KB and
IGROV-1 tumor-bearing mice, as compared to the combination with PMXsubther or untreated control mice.
PMXsubther protectedthekidneysfromundesiredside effectsof177Lu-EC0800(20MBq)byreducingtheabsorbed
radiation dose. Intact kidney function was shown by determination of plasma parameters and quantitative
single-photon emission computed tomography using 99mTc-DMSA. Our results confirmed the anticipated dual
role of PMX. Its unique features resulted in an improved antitumor effect of folate-based radionuclide therapy
and prevented undesired radio-nephrotoxicity. Mol Cancer Ther; 12(11); 2436–45. ꢀ2013 AACR.
The development of new targeting strategies for the
Introduction
treatment of further tumor types is of high interest and
would have a critical impact on the future management of
these cancer diseases. In this respect, the folate receptor is
an attractive target as it has been identified as specifically
associated with a variety of cancer types, such as ovarian,
endometrial, lung, brain, breast, and colorectal cancer (4,
5). The vitamin folic acid has been used as a targeting
ligand because it binds to the folate receptor with high
affinity followed by endocytotic internalization of the
therapeutic payload into cancer cells (6). While folic acid
conjugates of highly toxic chemotherapeutics have been
successfully used in clinical trials (7, 8), application of
therapeutic folic acid radioconjugates is currently being
developed in preclinical studies.
Targeted radionuclide therapy has shown impressive
results for the palliative treatment of several cancer dis-
eases. It is based on the use of particle-emitting radio-
isotopes (e.g., 177Lu, 90Y, 131I) in conjunction with tumor-
targeted biomolecules (e.g., peptides, antibodies; ref. 1). A
prominent example of a successfully used radiopharma-
ceutical in clinical routine are somatostatin-based radio-
peptides (e.g., 177Lu-DOTATATE, 90Y-DOTATOC) for
the treatment of neuroendocrine tumors (2). Moreover,
radiolabeled antibodies such as 90Y-Ibritumomab (Zeva-
lin) and 131I-tositumomab (Bexxar) are approved for the
treatment of non-Hodgkins lymphoma (3).
Substantial expression of the folate receptor in the
proximal tubule cells of the kidneys results in commonly
high and specific renal uptake of folate-based radio-
conjugates (9, 10). As a consequence there is an inherent
risk of damage to the kidneys by particle radiation. How-
ever, we have shown in several preclinical studies that
administration of the antifolate pemetrexed (PMX) re-
sulted in a tremendous reduction of the radiofolate’s
retention in the kidneys whereas accumulation in tumor
xenografts remained unaffected (11–13). The exact mech-
anism of this interaction is still not completely clear.
Authors' Affiliations: 1Center for Radiopharmaceutical Sciences ETH-
PSI-USZ, Paul Scherrer Institute, Villigen-PSI, Switzerland; 2Endocyte Inc.,
West Lafayette, Indiana
Note: Supplementary data for this article are available at Molecular Cancer
Therapeutics Online (http://mct.aacrjournals.org/).
€
Corresponding Author: Cristina Muller, Center for Radiopharmaceutical
Sciences ETH-PSI-USZ, Paul Scherrer Institute, 5232 Villigen-PSI, Switzer-
land. Phone: 41-56-310-44-54; Fax: 41-56-310-28-49; E-mail:
cristina.mueller@psi.ch
doi: 10.1158/1535-7163.MCT-13-0422-T
ꢀ2013 American Association for Cancer Research.
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Published OnlineFirst September 12, 2013; DOI: 10.1158/1535-7163.MCT-13-0422-T
177Lu-Folate Therapy in Combination with Pemetrexed
However, in numerous preclinical studies, we observed
an interrelation between the kidney reducing effect and
the time point of preinjected PMX (14) as well as the molar
amount of PMX and the folate radioconjugate, respective-
ly (15). However, the reduced kidney uptake of radio-
folates was not a result of PMX’s antifolate activity as the
effect was maintained even if PMX was applied in com-
bination with the antidote thymidine (16). These facts
suggest that PMX’s kidney reducing effect is based on a
competition among PMX and the folate radioconjugate for
folate receptor binding sites in the kidneys.
PMX is a multitargeted antifolate, which is clinically
approved for the treatment of pleural mesothelioma
and non–small cell lung cancer in combination with
cisplatin (17–19). Moreover, PMX is currently being
tested in a number of clinical trials for the treatment
of several other cancer types (reviewed in ref. 20),
among those is also ovarian cancer (21). PMX has
been used in combinations with gemcitabine, tyrosine
kinase inhibitors, antibodies, or even external radiation
(22–24). A combination of PMX with external radiother-
apy was based on the observation that PMX acts as a
radiosensitizing agent in variable types of cancer cells
in vitro and in vivo (25–28).
organisms and Cell Cultures (DSMZ). IGROV-1 cells
(human ovarian carcinoma cells) were a kind gift from
Dr. Gerrit Jansen (Department of Rheumatology, VU
University Medical Center, Amsterdam, the Nether-
lands). All 3 cell lines were authenticated by DMSZ based
on DNA profiling. KB and IGROV-1 cells are known to
express the folate receptor (31, 32), whereas folate receptor
negative PC-3 cells (33) were used as a negative control.
PC-3 cells were cultured in RPMI 1640 medium (Amimed)
whereas KB and IGROV-1 cells were cultured in folate-
free RPMI medium referred to as FFRPMI (without folic
acid, vitamin B12, and phenol red; Cell Culture Technol-
ogies GmbH). Cell culture media were supplemented
with 10% fetal calf serum, L-glutamine, and antibiotics
(penicillin/streptomycin/fungizone). Routine culture
treatment was conducted twice a week. Uptake and inter-
nalization studies of 177Lu-EC0800 as well as clonogenic
and MTT assays were conducted as previously reported
in the literature in detail in the Supplementary Methods
(29, 34).
MTT assay
Cell viability was assessed using an MTT assay (35). In
brief, KB, IGROV-1, and PC-3 cells were treated with
200 mL FFRPMI medium (without supplements) contain-
ing 177Lu-EC0800 (0.001–5.0 MBq/mL) and/or PMX
(0.01–100 mmol/L; pemetrexed, Alimta, LY231514; Eli
Lilly, Supplementary Fig. S1). The control cells were
incubated with FFRPMI medium only. After 4 hours
incubation at 37ꢀC, the cells were washed once with
200 mL PBS followed by addition of FFRPMI or RPMI
medium (with supplements). Cells were then allowed to
grow for 4 days at 37ꢀC without changing medium.
Analysis of the viability was conducted as previously
reported using an MTT reagent and a microplate reader
(Victor X3; Perkin Elmer; ref. 36). To quantify cell viability,
the absorbance of the test samples was expressed as
percentage of the absorbance of the control cell samples
(¼100%). Dose–response curves were analyzed using the
software GraphPad Prism (version 4.0). Inhibition of cell
viability was expressed as the half-maximal inhibitory
concentration of PMX (IC50 in mmol/L) and as the half-
maximal inhibitory activity concentration of 177Lu-
EC0800 (IAC50 in MBq/mL) by measuring dose–response
curves of PMX and 177Lu-EC0800. The dose–response
curves were used to determine the combination index
(CI) according to Chou and colleagues (Supplementary
Methods; ref. 37).
We hypothesized that PMX would have a dual role if it
was combined with therapeutic folate radioconjugates.
First, it was expected to prevent radionephropathy by
reducing the absorbed radiation dose of folate radio-
conjugates in the kidneys. Second, PMX was believed to
enhance the therapeutic efficacy of folate-based radio-
nuclide tumor therapy by its action as a chemotherapeutic
and radiosensitizing agent.
The goal of this study was to show the anticipated dual
role of PMX. For this purpose, we used an established
DOTA-folate conjugate (EC0800; ref. 29), which was
radiolabeled with 177Lu (T1/2 ¼ 6.7 days, Eav(b-): 134 keV,
E
(g): 113 keV, 208 keV). 177Lu-EC0800 was applied in
combination with PMX using 2 folate receptor positive
human cancer cells (KB and IGROV-1) in vitro and as
human tumor xenografts in athymic nude mice. More-
over, the protective effect of PMX on the kidneys to reduce
the absorbed radiation dose was investigated in non–
tumor-bearing mice.
Materials and Methods
Preparation of 177Lu-EC0800
The DOTA-folate conjugate (EC0800; ref. 29) was kindly
provided by Endocyte Inc. (Supplementary Fig. S1). The
radiosynthesis of 177Lu-EC0800 and the evaluation of its
stability were carried out as previously reported (Supple-
mentary Methods; ref. 30). It was shown that 177Lu-
EC0800 was stable (>95%) over at least 24 hours in PBS
and human blood plasma.
Animal studies
The in vivo experiments were approved by the local
veterinarian department and conducted in accordance
with the Swiss law of animal protection. Female athymic
nude mice (4–5 week-old CD-1 Foxn-1/nu; Charles River
Laboratories) were fed with a folate-deficient rodent diet
(Harlan Laboratories) starting 5 days before tumor cell
inoculation (38). For therapy experiments, endpoint cri-
teria were defined as (i) a tumor volume >1000 mm3, (ii)
Cell culture
KB cells (human cervical carcinoma cells, ACC-136),
and PC-3 (human prostate carcinoma cells, ACC-465)
were purchased from the German Collection of Micro-
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Reber et al.
body weight loss of >15%, (iii) active ulceration of the
tumor xenograft, or (iv) abnormal behavior of the mice
and signs of unease.
average tumor size reached a volume of ꢂ65 mm3 (KB
xenografts) and ꢂ40 mm3 (IGROV-1 xenografts). For each
study, 4 groups of 5 mice were injected with only PBS
(group A), with 2 ꢁ 0.8 mg PMX (i.e., PMXther, group B),
with 1 ꢁ 20 MBq 177Lu-EC0800 combined with 1 ꢁ 0.4 mg
PMX (i.e., PMXsubther, group C), and with 1 ꢁ 20 MBq
177Lu-EC0800 combined with 2 ꢁ 0.8 mg PMX (i.e.,
PMXther, group D).
Body weights and tumor volumes were measured 3
times a week. The tumor volume (V) was determined by
measuring 2 perpendicular diameters with a digital
caliper and calculated by using the formula V ¼ [0.5 ꢁ
(L ꢁ W2)], where L is the length of the tumor (large
diameter) and W is the width (small diameter; ref. 40).
The values were indicated as the average of the individual
relative tumor volumes (RTV ¼ Vx/V0; Vx ¼ volume at
day x, V0 ¼ volume at day 0) and of individual relative
body weight (RBW ¼ Wx/W0; Wx ¼ weight at day x; W0 ¼
weight at day 0). The therapeutic efficacy was expressed
as the percentage of tumor growth inhibition (%TGI ¼ 100
ꢃ (T/C ꢁ 100), where T is the mean RTV of the treated
mice and C is the mean RTV of untreated control mice at
day 17 when the first mouse in the control group was
euthanized. Tumor growth delay (TGD) was calculated
for the time that passed until the RTVs reached 4-fold the
value of day 0. The tumor growth delay index (TGDI) was
calculated as the ratio of TGDs among treated animals and
control animals (41).
Biodistribution study and dosimetric calculations
Biodistribution studies over 72 hours were conducted
as previously reported (Supplementary Methods and
Table S1; ref. 29). These datasets were used to estimate
the equivalent absorbed radiation dose to the tumor
xenografts and kidneys upon injection of 177Lu-EC0800
(Supplementary Methods). Based on the biodistribution
results the accumulation of radioactivity in KB and
IGROV-1 tumors was taken as equal. For estimation of
the kidney dose, it was assumed that in the case
of preinjected PMX kidney uptake was reduced to 25%
of control values.
Biodistribution studies conducted with 3H-PMX are
reported in the Supplementary Methods.
Investigation of potential radiotoxicity
Groups of 6 mice were injected with only PBS (group A),
with 177Lu-EC0800 (20 MBq, 1 nmol; group B) or with
177Lu-EC0800 (20 MBq, 1 nmol) and PMXsubther (0.4 mg;
group C). From day 21 after start of the therapy, the
animals were fed with a standard rodent diet. For study-
ing plasma parameters, blood was taken from the sub-
lingual vein collected in heparinized vials at day 50, 130,
and 180 or before euthanasia. Blood plasma parameters
such as creatinine, blood urea nitrogen, alkaline phospha-
tase, and total bilirubin were determined using a Fuji Dri-
Chem 40000i analyzer (Polymed Medical Center AG).
Quantitative single-photon emission computed tomogra-
phy (SPECT) to determine renal uptake of 99mTc-DMSA
in mice was conducted in week 3, 15, and 23 of the
experiment.
Statistical analysis
Statistics was conducted by using a t-test (Microsoft
Excel software). All analyses were 2-tailed and considered
as type 3 (2 sample unequal variance). A P-value of <0.05
was considered as statistically significant.
SPECT experiments were conducted with a 4-head
multiplexing multi-pinhole camera (NanoSPECT/CT;
Bioscan Inc.) using collimators of 4 ꢁ 9 holes of a diameter
of 1.4 mm. Dimercaptosuccinic acid (DMSA)-kits (Tech-
neScan) were purchased from Mallinckrodt Inc. The
99mTc-radiolabeling was conducted according to the
instructions of the manufacturer using 99mTc-per-
technetate, which was freshly eluted from a 99mTc-gener-
ator (Mallinckrodt Inc.). SPECT images of the kidneys
(30-mm scan length) were acquired 2 hours after injection
of 99mTc-DMSA (ꢂ30 MBq/mouse). After acquisition,
SPECT data were reconstructed iteratively with HiSPECT
software (version 1.4.3049; Scivis GmbH) using a g-energy
of 140 keV for 99mTc. Quantification of renal uptake of
99mTc-DMSA was carried out as previously reported
using InVivoScope postprocessing software (version
2.0; Bioscan Inc.; ref. 39).
Results
Cancer cell lines
The human cervical KB cancer cell line is a subclone of
HeLa cells (42), known to express the folate receptor at
very high levels. IGROV-1 cells are human ovarian cancer
cells that express the folate receptor at a somewhat lower
level than KB cells (12, 31, 36). The human prostate cancer
cell line PC-3 does not express the folate receptor and was
used as a negative control. These facts were confirmed in
uptake/internalization studies of 177Lu-EC0800 (Supple-
mentary Fig. S2).
Cell survival upon exposure to 177Lu-EC0800
Clonogenic assays revealed plating efficiencies of 11%,
14%, 18% and 16%, for KB, IGROV-1, and PC-3 cells. At a
radioactivity concentration of 1.0 MBq/mL 177Lu-EC0800
(16 nmol/L) with a 4 hours exposure, the survival fraction
of KB and IGROV-1 cells was 0.12 and 0.43, respectively.
The survival fraction of folate receptor negative PC-3 cells
was still 0.98, even after the treatment with a 5-fold higher
radioactivity concentration. Reduction of cell survival
was completely suppressed by preincubating folate
Therapy studies in tumor-bearing mice
Twenty mice wereinoculated with KB tumorcells (4.5 ꢁ
106 in 100 mL PBS, model I) and another 20 mice were
inoculated with IGROV-1 tumor cells (6.0 ꢁ 106 in 100 mL
PBS, model II). At the start of the therapy 4 days later, the
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177Lu-Folate Therapy in Combination with Pemetrexed
Combination index
The interactions between 177Lu-EC0800 and PMX were
calculated according to the results obtained with KB and
IGROV-1 cells, which were exposed to 177Lu-EC0800 and
PMX as single agents or simultaneously. The concentra-
tions of the test agents, alone and in combination, required
to reduce cell viability to 55% and 70% of controls were
determined. All calculations revealed values of the CI
below 0.8, indicating a synergistic effect between 177Lu-
EC0800 and PMX (Supplementary Table S2).
Biodistribution studies
Biodistribution studies in KB and IGROV-1 tumor-
bearing mice showed a relatively high uptake of 177Lu-
EC0800 in tumor xenografts [KB: 5.94 ꢄ 1.20% ID/g and
IGROV-1: 6.58 ꢄ 1.50% ID/g; 4 hours past injection (p.i)],
and a ꢂ10-fold higher accumulation in the folate receptor
positive kidneys. PMX treatment reduced renal uptake of
177Lu-EC0800 up to 7-fold, while simultaneously allowing
for greater tumor uptake (Table 1). Dosimetric estimation
revealed a dose of 0.38 Gy/MBq to the tumor xenografts
and 4.84 Gy/MBq to the kidneys if 177Lu-EC0800 was
applied as a single agent. Under the assumption of a 4-fold
reduced renal uptake of 177Lu-EC0800 in combination
with PMX, the kidney dose was reduced to 1.21 Gy/MBq
whereas the tumor dose remained unaffected (Supple-
mentary Methods and Fig. S4).
Short after injection of 3H-PMX, the uptake in the
kidneys was relatively high (ꢂ2.7% ID/g, 1 hour p.i.) but
a significant washout was observed within the following
hour (ꢂ0.8% ID/g, 2 hours p.i.). In the tumor tissue
3
accumulation of H-PMX was low (ꢂ1.1% ID/g, 1 hour
p.i.) but more constant (0.6% ID/g, 2 hours p.i.) over time
(Supplementary Methods and Fig. S5).
Investigation of potential radiotoxicity
In a separate study, radiotoxicity of 177Lu-EC0800 and
the kidney protective effect of a subtherapeutic amount of
PMX (0.4 mg) were investigated. Nontumor-bearing nude
mice were monitored over 6 months. The kidney dose of
177Lu-EC0800 (20 MBq/mouse) was ꢂ97 Gy (group B). If
PMXsubther was preinjected, the kidney dose was signif-
icantly reduced to ꢂ24 Gy (group C). At day 50 of the
study, plasma parameters of treated mice (groups B/C)
were in the same range as those of control mice (group A).
However, at day 130, levels of blood urea nitrogen, alka-
line phosphatase, and total bilirubin from mice treated
with 177Lu-EC0800 (group B) differed significantly from
those of control mice (group A). The values obtained from
mice of group C were in the same range as the values
from mice of group A. Determination of blood plasma
parameters at day 180 showed the same result as found at
day 130 (Table 2).
Figure 1. A, survival of folate receptor–positive KB and IGROV-1 cells
and folate receptor–negative PC-3 cells upon exposure to
(1 MBq/mL; 16 nmol/L) in the presence and absence of excess folic acid
(FA). B and C, dose–response curves of KB and IGROV-1 tumor cells
incubated with variable activity concentrations of 177Lu-EC0800
(indicated as 177Lu) and PMX.
177Lu-EC0800
receptor positive tumor cells with excess folic acid to block
folate receptors (Fig. 1A; Supplementary Fig. S3).
Cell viability upon exposure of 177Lu-EC0800
combined with PMX
MTT assays were conducted to determine IAC50 and
IC50 values of 177Lu-EC0800 and PMX. The inhibition of
cell viability was found to be dependent on the concen-
tration of PMX in all cell lines. The IC50 value of PMX
amounted to 1.22 ꢄ 0.13 mmol/L and 0.93 ꢄ 0.17 mmol/L
for KB and IGROV-1 cells, respectively. The IAC50-values
of 177Lu-EC0800 revealed activity concentrations of 0.054
ꢄ 0.004 MBq/mL for KB and 0.83 ꢄ 0.08 MBq/mL for
IGROV-1 tumor cells (Fig. 1B and C).
The extent of accumulated 99mTc-DMSA in the kidneys
is a measure for tubular function (43). It has previously
been used as a valuable in vivo tool for monitoring kidney
function during radionuclide therapy (39). In week 3,
baseline measurements of 99mTc-DMSA uptake in the
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Reber et al.
Table 1. Biodistribution data 4 hours after injection of 177Lu-EC0800 (3 MBq, 1 nmol) in KB (model I)
and IGROV-1 tumor-bearing nude mice (model II)
177Lu-EC0800 (model I)
a
b
Control
PMXsubther
PMXther
Blood
Liver
Kidneys
Tumor
Tumor-to-blood
Tumor-to-liver
Tumor-to-kidney
0.08 ꢄ 0.01
6.61 ꢄ 1.04
51.0 ꢄ 6.6
0.06 ꢄ 0.01
4.28 ꢄ 0.69
9.66 ꢄ 1.14
7.83 ꢄ 0.98
131.1 ꢄ 24.6
1.88 ꢄ 0.41
0.86 ꢄ 0.14
0.06 ꢄ 0.01
3.46 ꢄ 0.38
8.12 ꢄ 0.88
9.01 ꢄ 1.60
161.7 ꢄ 17.3
2.64 ꢄ 0.65
1.11 ꢄ 0.16
5.94 ꢄ 1.20
71.6 ꢄ 19.2
0.91 ꢄ 0.22
0.12 ꢄ 0.02
177Lu-EC0800 (model II)
a
b
Control
PMXsubther
PMXther
Blood
Liver
Kidneys
Tumor
Tumor-to-blood
Tumor-to-liver
Tumor-to-kidney
0.16 ꢄ 0.04
7.60 ꢄ 2.04
67.0 ꢄ 13.4
6.58 ꢄ 1.50
42.7 ꢄ 7.8
0.08 ꢄ 0.00
2.75 ꢄ 0.19
11.4 ꢄ 1.2
0.08 ꢄ 0.02
2.37 ꢄ 0.68
9.30 ꢄ 1.96
9.55 ꢄ 0.88
127.9 ꢄ 40.6
4.32 ꢄ 1.48
1.04 ꢄ 0.54
9.30 ꢄ 1.96
122.7 ꢄ 28.0
3.40 ꢄ 0.78
0.81 ꢄ 0.13
0.88 ꢄ 0.16
0.10 ꢄ 0.02
NOTE: Data are presented as average ꢄ SD (n ¼ 3).
aPMXsubther: 0.4 mg per mouse, injected 1 hour before 177Lu-EC0800.
bPMXther: 0.8 mg per mouse, injected 1 hour before 177Lu-EC0800.
kidneys showed no significant difference among groups B
and C from control mice of group A (Fig. 2A). However, in
week 15 the average % ID per kidney of mice treated with
177Lu-EC0800 only (group B: 4.37 ꢄ 2.6% ID/kidney, 2
hours p.i., P < 0.005) was significantly lower than in
control mice (group A: 11.74 ꢄ 0.7% ID/kidney, 2 hours
Table 2. Plasma parameters of group A (PBS), group B (20 MBq of 177Lu-EC0800), and group C (20 MBq of
177Lu-EC0800 and 0.4 mg of PMX)
Group C
Group A
PBS
Group B
177Lu-EC0800 and
PMXsubther
Day
177Lu-EC0800
CRE (mmol/L)
BUN (mmol/L)
ALP (U/L)
50
130
180a
21 ꢄ 3.4
28 ꢄ 12
19 ꢄ 0.7
8.5 ꢄ 0.9
9.7 ꢄ 0.6
8.8 ꢄ 0.6
78 ꢄ 10
54 ꢄ 16
58 ꢄ 14
19 ꢄ 2.1
58 ꢄ 43
<18
<18
21 ꢄ 0.7
10.0 ꢄ 1.1
8.61 ꢄ 1.1
9.09 ꢄ 1.4
114 ꢄ 62b
50
130
180a
11.8 ꢄ 2.2b
35.0 ꢄ 9.4c
>49.98c
50
130
180a
83 ꢄ 20
132 ꢄ 42c
123 ꢄ 55b
70 ꢄ 8
71 ꢄ 18
67 ꢄ 23
TBIL (mmol/L)
50
130
180a
10 ꢄ 1.3
12 ꢄ 1.9
12 ꢄ 3.2
8 ꢄ 1.0
24 ꢄ 11b
36 ꢄ 1.9c
11 ꢄ 1.7
12 ꢄ 4.5
12 ꢄ 5.0
Abbreviations: CRE, creatinine; BUN, blood urea nitrogen; ALP, alkaline phosphatase; TBIL, total bilirubin.
aGroup B terminal blood sampling.
bP < 0.05.
cP < 0.005.
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177Lu-Folate Therapy in Combination with Pemetrexed
Figure 2. A–C, the average %ID
per kidney in week 3, 15, and 23
99mTc-DMSA in
after injection of
untreated control mice (group A),
mice treated with 177Lu-EC0800
(group B), and mice treated with
177Lu-EC0800 and PMXsubther
(group C; ꢅꢅ, P < 0.005). D, graphs
of the relative body weights of mice
from each group over the whole
time of 6 months. The lowest
tolerable body weight is indicated
at a relative value of 0.85. E, SPECT
images of kidneys after injection of
99mTc-DMSA of one representative
mouse of each group in week 23.
p.i.). Renal uptake of 99mTc-DMSA in mice that received
PMXsubther in addition to 177Lu-EC0800 was comparable
(group C: 10.58 ꢄ 1.8% ID/kidney, 2 hours p.i., P ¼ 0.2)
with the value obtained from control animals (Fig. 2B). In
week 23 of follow-up, renal accumulation of 99mTc-DMSA
inmiceofgroup B haddropped to 1.38ꢄ 0.16% ID/kidney
2 hours p.i. (P < 0.005), whereas in group C the uptake was
still in same range as found for control animals of group A
(P ¼ 0.6; Fig. 2C).
mouse reached the endpoint criterion at day 17. For
model I, the average RTV in mice treated with PMXther
(groupB:9.8ꢄ 4.1;P ¼ 0.90)and inmicetreatedwith 177Lu-
EC0800 and PMXsubther (group C: 8.3 ꢄ 3.5, P ¼ 0.21) were
not significantly different from the average RTV of control
mice (group A: 10.0 ꢄ 2.7) at day 17 (Table 3). However, the
combined application of 177Lu-EC0800 with PMXther
resulted in a significant decrease of the average RTV
(group D: 5.0 ꢄ 2.2; P < 0.0001; Fig. 3B). For tumor model
II, the average RTV of PMX-treated mice (group B: 27.9 ꢄ
11.8; P ¼ 0.73) was not significantly reduced compared to
control mice (group A: 30.0 ꢄ 14.9) at the same time point
(Table 3). However, a significant reduction of the average
RTV was observed in both groups of mice treated with
177Lu-EC0800 (group C: 7.3 ꢄ 3.9, P ¼ 0.0006; group D:
2.0 ꢄ 1.7, P ¼ 0.0002; Fig. 3C). Monitoring of the body
weight revealed slight weight gain over time in model I
anda largely constant body weight in model II(Fig. 3D and
E). In contrast, pronounced loss of body weight was
observed in group B mice of both tumor models (which
received PMXther only). Importantly, the average survival
time was increased 75–100% in group D mice from both
tumor models compared to group A mice (Table 3).
Constant body weight loss was observed in mice treated
with 177Lu-EC0800 (group B) from about day 70 and
thereafter. In all cases of group B, the endpoint criterion
that required euthanasia was reached before day 180.
However, mice that had received PMXsubther before the
injection of 177Lu-EC0800 (group C) showed body weight
gain similar to the untreated controls (group A).
In vivo tumor therapy studies
The injection protocol of the therapy study with KB and
IGROV-1 tumor-bearing mice is shown in Fig. 3A. PMX
was applied either in a subtherapeutic dose of 0.4 mg
(PMXsubther) or at a therapeutic dose of 2 ꢁ 0.8 mg
(PMXther) corresponding to 80% of the maximal tolerated
dose (MTD). The MTD of PMX was previously deter-
mined in mice under the experimental conditions of a
folate-free diet and revealed a dose of 2 ꢁ 1 mg per mouse
(body weight ꢂ 25 g) with a time lag of 1 week (Supple-
mentary Methods and Fig. S6; ref. 16).
Discussion
Combining anticancer therapies is a strategy to broaden
thetherapeutic indexbytakinganadvantageofadditive or
synergistic antitumor effects and by reducing undesired
side effects. This study addressed the question of whether
In both tumor models (I and II), constant tumor growth
was observed in control mice (group A) where the first
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Reber et al.
Figure 3. A, injection scheme of the
in vivo therapy studies conducted
in KB (model I) and IGROV-1
tumor-bearing mice (model II). B,
graphs of the mean relative tumor
volumes of KB tumor xenografts
(model I). C, graphs of mean
relative tumor volumes of IGROV-1
xenografts (model II). D, graphs of
the mean relative body weights of
KB tumor-bearing mice (model I).
E, graphs of the mean relative body
weight of IGROV-1 tumor-bearing
mice (model II).
PMX contributes to the antitumor effect of radiofolates
and prevents the risk of radionephropathy. In vitro the
clonogenic potential of KB and IGROV-1 tumor cells was
reduced upon exposure to 177Lu-EC0800 in a concentra-
tion-dependent manner (Fig. 1). Moreover it was proven
that this effect was specifically folate receptor mediated.
It was more pronounced in KB cells, which express the
folate receptor at higher levels than IGROV-1 cells, and it
was abolished if the cells were coincubated with excess
folic acid to block folate receptor binding of 177Lu-EC0800.
Inhibition of cell viability through application of 177Lu-
EC0800 was enhanced if cells were coincubated with
PMX (Fig. 1). Determination of the combination indices
at different drug concentrations revealed that 177Lu-
EC0800 and PMX provided synergistic inhibitory effects
on the viability of both tumor cell lines. It was observed
that incubation of the cancer cells with PMX resulted in an
accumulation of the cells in the G1–S boundary or early
S phase as previously reported (Supplementary Meth-
ods and Fig. S7; refs. 25 and 44). However, exposure of
cells to 177Lu-EC0800 showed a cell-cycle arrest in the
G2 –M phase, which is a common phenomenon in
eukaryotic cells exposed to ionizing radiation (45).
However, if the cancer cells were simultaneously
exposed to 177Lu-EC0800 and PMX the cell-cycle arrest
in G2–M phase was abrogated. Notably, the disruption
of the radiation-induced G2-checkpoint by chemo-
therapeutic agents (e.g., protein kinase inhibitors)
was previously shown to sensitize cancer cells to radi-
ation-induced apoptosis and cell death (46–48). This
mechanism might also have been responsible for
PMX-induced radiosensitization of KB and IGROV-1
cells. An increased apoptotic cell fraction was measured
if the cells were treated with 177Lu-EC0800 and PMX
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Molecular Cancer Therapeutics
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177Lu-Folate Therapy in Combination with Pemetrexed
Table 3. Results of the therapy studies with KB and IGROV-1 tumor-bearing mice using 177Lu-EC0800
and/or PMX
Model I: KB tumor xenografts
Radioactivity
(MBq)
Average survival
time (%)
Group
PMX (mg)
RTV day 17
TGI (%)
TGDI4
A
B
C
D
–
–
10.0 ꢄ 2.7
9.8 ꢄ 4.1
8.3 ꢄ 3.5
5.0 ꢄ 2.2
–
1.0
1.2
1.4
1.9
–
2 ꢁ 0.8(ther)
1 ꢁ 0.4(subther)
2 ꢁ 0.8(ther)
–
2.1
16.9
50.4
þ22.5%
þ50.0%
þ75.0%
1 ꢁ 20
1 ꢁ 20
Model II: IGROV-1 tumor xenografts
Radioactivity
(MBq)
Average survival
time (%)
Group
PMX (mg)
RTV day 17
TGI (%)
TGDI4
A
B
C
D
–
–
30.0 ꢄ 14.9
27.9 ꢄ 11.8
7.3 ꢄ 3.9
0.0
7.1
75.8
93.2
1.0
1.8
2.6
4.0
–
2 ꢁ 0.8(ther)
1 ꢁ 0.4(subther)
2 ꢁ 0.8(ther)
–
þ0%
þ63.0%
þ100%
1 ꢁ 20
1 ꢁ 20
2.0 ꢄ 1.7
compared to the application of each of these therapeu-
tics alone (Supplementary Methods and Fig. S8).
In vivo the preinjection of PMX improved the tissue
distribution of 177Lu-EC0800 dramatically by increasing
the tumor-to-kidney ratio (Table 1). In an attempt to
understand the underlying mechanism, the uptake of
3H-PMX was determined in KB tumors and kidneys of
mice. In the kidneys, we found a relatively high uptake
PMX (2 ꢁ 0.8 mg, corresponding to 80% of the MTD;
ref. 16). Therapy studies were conducted with KB (model
I) and IGROV-1 tumor-bearing mice (model II) using
177Lu-EC0800 combined with either subtherapeutic
(0.4 mg/mouse) or therapeutic doses (e.g., 2 ꢁ 0.8 mg)
of PMX. In both models, tumor growth delay was
observed after application of 177Lu-EC0800. Application
of PMXther alone showed only minor inhibitory effects on
growth of these tumor types. However, PMXther was able
to enhance the anticancer effect of 177Lu-EC0800 against
both KB and IGROV-1 tumor xenografts. Also, an
increased survival time was achieved if 177Lu-EC0800 and
PMX were combined compared to the result obtained
with each of these agents applied as monotherapy.
With this study we were able to show the proposed
dual effect of PMX in combination with folate receptor
targeted radionuclide therapy using 177Lu-EC0800. On one
hand, PMX at subtherapeutic and therapeutic amounts
effectively reduced renal uptake of 177Lu-EC0800 and
therewith prevented long-term radionephropathy (Table
1, Fig. 2). On the other hand, the application of PMXther
enhanced the tumor growth delay induced by 177Lu-
EC0800. The interplay of the proposed drug combination
is absolutely unique. Therefore, we believe that combining
PMXwith 177Lu-folatetherapywarrantsfurtherpreclinical
investigations. Assuming the kidney-reducing effect of
PMX could be confirmed also in man, the combined
application of therapeutic radiofolates and PMX has a
potential translational impact. Such a therapy protocol
would be particularly interesting for the treatment of
non–small cell lung cancer, which frequently shows folate
receptor overexpression and for which PMX is a Food and
Drug Administration-approved indication.
3
1 hour p.i. of H-PMX, which was quickly cleared over
time (Supplementary Fig. S5). These findings, together
with the fact that PMX has a high affinity for the folate
receptor (49), support our hypothesis of a competition
among PMX and 177Lu-EC0800 for folate receptor binding
sites in the kidneys if PMX was injected about 1 hour
before the radiofolate. In the tumor tissue, uptake of 3H-
PMX was low but retained over time. It has been reported
previously that the tumor uptake of PMX is mediated
primarily through carriers such as the reduced folate
carrier and the proton-coupled folate transporter, where-
as folate receptors play a minor role (50). This may explain
the fact that PMX does not compete with the folate recep-
tor–mediated accumulation of 177Lu-EC0800 in tumor
xenografts.
For the first time we were able to show in this study
that PMX prevents damage to the kidneys by reducing
renal accumulation of 177Lu-EC0800. Analysis of
plasma parameters and the results of SPECT studies
using 99mTc-DMSA consistently confirmed normal
kidney function in 177Lu-EC0800-treated mice that had
received a subtherapeutic amount of PMX. These findings
unambiguously confirmed the beneficial role of PMX
to prevent radionephropathy of radiofolate therapy.
Based on the in vitro results showing a synergistic effect
of PMX and 177Lu-EC0800 on the viability of tumor cells, it
is likely that the anticancer effect of 177Lu-EC0800 would
be enhanced by coapplication of therapeutic amounts of
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
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2443
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Reber et al.
authors are grateful for technical assistance of the experiments by
N. Romano.
Authors' Contributions
€
Conception and design: J. Reber, C. Muller
€
Development of methodology: J. Reber, C. Muller
Acquisition of data (provided animals, acquired and managed patients,
Grant Support
€
provided facilities, etc.): J. Reber, S. Haller, C. Muller
Analysis and interpretation of data (e.g., statistical analysis, biostatis-
€
C. Muller was financially supported by the Swiss National Science
Foundation (Ambizione, Grants PZ00P3_121772 and PZ00P3_138834),
S. Haller was supported by the Swiss Cancer League (KLS-02762-02-
2011), and J. Reber was supported by COST-BM0607 (C08.0026).
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
€
tics, computational analysis): J. Reber, C.P. Leamon, C. Muller
Writing, review, and/or revision of the manuscript: J. Reber, S. Haller,
€
C.P. Leamon, C. Muller
Administrative, technical, or material support (i.e., reporting or orga-
nizing data, constructing databases): J. Reber, S. Haller
€
Study supervision: C. Muller
Acknowledgments
The authors thank Prof. R. Schibli for helpful advice with regard
to the design of this study and for reviewing the manuscript. The
Received May 30, 2013; revised August 29, 2013; accepted September 8,
2013; published OnlineFirst September 12, 2013.
References
1. Zoller F, Eisenhut M, Haberkorn U, Mier W. Endoradiotherapy in cancer
17. Vogelzang NJ, Rusthoven JJ, Symanowski J, Denham C, Kaukel E,
Ruffie P, et al. Phase III study of pemetrexed in combination with
cisplatin versus cisplatin alone in patients with malignant pleural
mesothelioma. J Clin Oncol 2003;21:2636–44.
18. Hazarika M, White RM, Johnson JR, Pazdur R. FDA drug approval
summaries: pemetrexed (Alimta). Oncologist 2004;9:482–8.
19. Rollins KD, Lindley C. Pemetrexed: a multitargeted antifolate. Clin Ther
2005;27:1343–82.
treatment—basic concepts and future trends. Eur
2009;625:55–62.
J Pharmacol
2. Kam BL, Teunissen JJ, Krenning EP, de Herder WW, Khan S, van Vliet
EI, et al. Lutetium-labelled peptides for therapy of neuroendocrine
tumours. Eur J Nucl Med Mol Imaging 2012;39 Suppl 1:S103–12.
3. Tomblyn M. Radioimmunotherapy for B-cell non-Hodgkin lympho-
mas. Cancer Contr 2012;19:196–203.
4. Parker N, Turk MJ, Westrick E, Lewis JD, Low PS, Leamon CP. Folate
receptor expression in carcinomas and normal tissues determined by a
quantitative radioligand binding assay. Anal Biochem 2005;338:
284–93.
5. Low PS, Kularatne SA. Folate-targeted therapeutic and imaging
agents for cancer. Curr Opin Chem Biol 2009;13:256–62.
6. Salazar MD, Ratnam M. The folate receptor: what does it promise in
tissue-targeted therapeutics? Cancer Metastasis Rev 2007;26:141–52.
7. Dosio F, Milla P, Cattel L. EC-145, a folate-targeted Vinca alkaloid
conjugate for the potential treatment of folate receptor-expressing
cancers. Curr Opin Investig Drugs 2010;11:1424–33.
8. Pribble P, Edelman MJ. EC145: a novel targeted agent for adenocar-
cinoma of the lung. Expert Opin Investig Drugs 2012;21:755–61.
9. Holm J, Hansen SI, Hoiermadsen M, Bostad L. A high-affinity folate
binding-protein in proximal tubule cells of human kidney. Kidney Int
1992;41:50–5.
10. Sandoval RM, Kennedy MD, Low PS, Molitoris BA. Uptake and
trafficking of fluorescent conjugates of folic acid in intact kidney
determined using intravital two-photon microscopy. Am J Physiol Cell
Physiol 2004;287:C517–26.
20. Hanauske AR, Chen V, Paoletti P, Niyikiza C. Pemetrexed disodium: a
novel antifolate clinically active against multiple solid tumors. Oncolo-
gist 2001;6:363–73.
21. Morotti M, Valenzano Menada M, Venturini PL, Mammoliti S, Ferrero S.
Pemetrexed disodium in ovarian cancer treatment. Expert Opin Inves-
tig Drugs 2012;21:437–49.
22. Monnerat C, Le Chevalier T. Review of the pemetrexed and gemcita-
bine combination in patients with advanced-stage non-small cell lung
cancer. Ann Oncol 2006;17 Suppl 5:v86–90.
23. Govindan R, Bogart J, Stinchcombe T, Wang X, Hodgson L, Kratzke R,
et al. Randomized phase II study of pemetrexed, carboplatin, and
thoracic radiation with or without cetuximab in patients with locally
advanced unresectable non-small-cell lung cancer: cancer and Leu-
kemia Group B trial 30407. J Clin Oncol 2011;29:3120–5.
24. Seiwert TY, Connell PP, Mauer AM, Hoffman PC, George CM, Szeto L,
et al. A phase I study of pemetrexed, carboplatin, and concurrent
radiotherapy in patients with locally advanced or metastatic non-small
cell lung or esophageal cancer. Clin Cancer Res 2007;13:515–22.
25. Bischof M, Weber KJ, Blatter J, Wannenmacher M, Latz D. Interaction
of pemetrexed disodium (ALIMTA, multitargeted antifolate) and irra-
diation in vitro. Int J Radiat Oncol Biol Phys 2002;52:1381–8.
26. Huber PE, Bischof M, Jenne J, Heiland S, Peschke P, Saffrich R,
et al. Trimodal cancer treatment: beneficial effects of combined
antiangiogenesis, radiation, and chemotherapy. Cancer Res 2005;
65:3643–55.
€
11. Muller C, Reddy JA, Leamon CP, Schibli R. Effects of the antifolates
pemetrexed and CB3717 on the tissue distribution of 99mTc-EC20 in
xenografted and syngeneic tumor-bearing mice. Mol Pharm 2010;7:
597–604.
€
12. Muller C, Schibli R, Krenning EP, de Jong M. Pemetrexed improves
tumor selectivity of 111In-DTPA-folate in mice with folate receptor-
27. Yoshida D, Ebara T, Sato Y, Kaminuma T, Takahashi T, Asao T, et al.
Interaction of radiation and pemetrexed on a human malignant meso-
thelioma cell line in vitro. Anticancer Res 2011;31:2847–51.
28. Shewach DS, Lawrence TS. Antimetabolite radiosensitizers. J Clin
Oncol 2007;25:4043–50.
positive ovarian cancer. J Nucl Med 2008;49:623–9.
€
13. Muller C, Mindt TL, de Jong M, Schibli R. Evaluation of a novel
radiofolate in tumour-bearing mice: promising prospects for folate-
based radionuclide therapy. Eur J Nucl Med Mol Imaging 2009;36:
938–46.
€
29. Muller C, Vlahov IR, Santhapuram HK, Leamon CP, Schibli R. Tumor
14. Muller C, Bruhlmeier M, Schubiger AP, Schibli R. Effects of antifolate
drugs on the cellular uptake of radiofolates in vitro and in vivo. J Nucl
Med 2006;47:2057–64.
targeting using 67Ga-DOTA-Bz-folate—investigations of methods to
improve the tissue distribution of radiofolates. Nucl Med Biol 2011;
38:715–23.
€
€
€
€
15. Muller C, Schibli R, Forrer F, Krenning EP, de Jong M. Dose-dependent
30. Muller C, Struthers H, Winiger C, Zhernosekov K, Schibli R. DOTA
effects of (anti)folate preinjection on 99mTc-radiofolate uptake in
conjugate with an albumin-binding entity enables the first folic acid-
tumors and kidneys. Nucl Med Biol 2007;34:603–8.
targeted 177Lu-radionuclide tumor therapy in mice.
J Nucl Med
€
16. Muller C, Reber J, Schlup C, Leamon CP, Schibli R. In vitro and in vivo
2013;54:124–31.
evaluation of an innocuous drug cocktail to improve the quality of folic
acid targeted nuclear imaging in preclinical research. Mol Pharm 2013;
10:967–74.
31. Weitman SD, Lark RH, Coney LR, Fort DW, Frasca V, Zurawski VR,
et al. Distribution of the folate receptor GP38 in normal and malignant
cell lines and tissues. Cancer Res 1992;52:3396–401.
Mol Cancer Ther; 12(11) November 2013
Molecular Cancer Therapeutics
2444
Downloaded from mct.aacrjournals.org on March 8, 2015. © 2013 American Association for Cancer Research.

Published OnlineFirst September 12, 2013; DOI: 10.1158/1535-7163.MCT-13-0422-T
177Lu-Folate Therapy in Combination with Pemetrexed
€
32. Muller C, Schubiger PA, Schibli R. In vitro and in vivo targeting of
human interferon-a or interferon-b with ifosfamide. Oncogene 2002;
21:7700–9.
42. Jiang L, Zeng X, Wang Z, Chen Q. Cell line cross-contamination: KB is
different folate receptor-positive cancer cell lines with a novel
99mTc-radiofolate tracer. Eur J Nucl Med Mol Imaging 2006;33:
1162–70.
not an oral squamous cell carcinoma cell line. Eur
J Oral Sci
33. Hattori Y, Maitani Y. Folate-linked nanoparticle-mediated suicide gene
therapy in human prostate cancer and nasopharyngeal cancer with
herpes simplex virus thymidine kinase. Cancer Gene Ther 2005;12:
796–809.
34. Franken NA, Rodermond HM, Stap J, Haveman J, van Bree C.
Clonogenic assay of cells in vitro. Nat Protoc 2006;1:2315–9.
35. Mosmann T. Rapid colorimetric assay for cellular growth and survival:
application to proliferation and cytotoxicity assays. J Immunol Meth-
ods 1983;65:55–63.
2009;117:90–1.
43. Weyer K, Nielsen R, Petersen SV, Christensen EI, Rehling M, Birn H.
Renal uptake of 99mTc-dimercaptosuccinic acid is dependent on
normal proximal tubule receptor-mediated endocytosis. J Nucl Med
2013;54:159–65.
44. Tonkinson JL, Worzalla JF, Teng CH, Mendelsohn LG. Cell cycle
modulation by a multitargeted antifolate, LY231514, increases the
cytotoxicity and antitumor activity of gemcitabine in HT29 colon
carcinoma. Cancer Res 1999;59:3671–6.
€
36. Reber J, Struthers H, Betzel T, Hohn A, Schibli R, Muller C. Radio-
45. Maity A, McKenna WG, Muschel RJ. The molecular basis for cell cycle
delays following ionizing radiation: a review. Radiother Oncol 1994;
31:1–13.
46. Yao SL, Akhtar AJ, McKenna KA, Bedi GC, Sidransky D, Mabry M, et al.
Selective radiosensitization of p53-deficient cells by caffeine-mediat-
ed activation of p34cdc2 kinase. Nat Med 1996;2:1140–3.
47. Busby EC, Leistritz DF, Abraham RT, Karnitz LM, Sarkaria JN. The
radiosensitizing agent 7-hydroxystaurosporine (UCN-01) inhibits the
DNA damage checkpoint kinase hChk1. Cancer Res 2000;60:
2108–12.
iodinated folic acid conjugates: evaluation of a valuable concept
to improve tumor-to-background contrast. Mol Pharm 2012;9:
1213–21.
37. Chou TC. Theoretical basis, experimental design, and computerized
simulation of synergism and antagonism in drug combination studies.
Pharmacol Rev 2006;58:621–81.
38. Mathias CJ, Wang S, Lee RJ, Waters DJ, Low PS, Green MA. Tumor-
selective radiopharmaceutical targeting via receptor-mediated endo-
cytosis of gallium-67-deferoxamine-folate.
J Nucl Med 1996;37:
1003–8.
48. Wang Y, Li J, Booher RN, Kraker A, Lawrence T, Leopold WR, et al.
Radiosensitization of p53 mutant cells by PD0166285, a novel G2
checkpoint abrogator. Cancer Res 2001;61:8211–7.
39. Forrer F, Valkema R, Bernard B, Schramm NU, Hoppin JW, Rolleman E,
et al. In vivo radionuclide uptake quantification using a multi-pinhole
SPECT system to predict renal function in small animals. Eur J Nucl
Med Mol Imaging 2006;33:1214–7.
40. Leamon CP, Reddy JA, Vlahov IR, Westrick E, Parker N, Nicoson JS,
et al. Comparative preclinical activity of the folate-targeted Vinca
alkaloid conjugates EC140 and EC145. Int
1585–92.
41. Sanceau J, Poupon MF, Delattre O, Sastre-Garau X, Wietzerbin J.
49. Westerhof GR, Schornagel JH, Kathmann I, Jackman AL, Rosowsky A,
Forsch RA, et al. Carrier- and receptor-mediated transport of folate
antagonists targeting folate-dependent enzymes: correlates of molec-
ular-structure and biological activity. Mol Pharmacol 1995;48:459–71.
50. Zhao R, Qiu A, Tsai E, Jansen M, Akabas MH, Goldman ID. The proton-
coupled folate transporter: impact on pemetrexed transport and on
antifolates activities compared with the reduced folate carrier. Mol
Pharmacol 2008;74:854–62.
J Cancer 2007;121:
Strong inhibition of Ewing tumor xenograft growth by combination of
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Published OnlineFirst September 12, 2013; DOI: 10.1158/1535-7163.MCT-13-0422-T
177Lu-EC0800 Combined with the Antifolate Pemetrexed:
Preclinical Pilot Study of Folate Receptor Targeted Radionuclide
Tumor Therapy
Josefine Reber, Stephanie Haller, Christopher P. Leamon, et al.
Mol Cancer Ther 2013;12:2436-2445. Published OnlineFirst September 12, 2013.
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