Radioiodinated folic acid conjugates: Evaluation of a valuable concept to improve tumor-to-background contrast

Article abstract of DOI:10.1021/mp200511t

Folic acid radioconjugates can be used for targeting folate receptor positive (FR⁺) tumors. However, the high renal uptake of radiofolates is a drawback of this strategy, particularly with respect to a therapeutic application due to the risk of damage to the kidneys by particle radiation. The goal of this study was to develop and evaluate radioiodinated folate conjugates as a novel class of folate-based radiopharmaceuticals potentially suitable for therapeutic application. Two different folic acid conjugates, tyrosine-folate (1) and tyrosine-click-folate (3), were synthesized and radioiodinated using the Iodogen method resulting in [¹²⁵I]-2 and [^125/131I]-4. Both radiofolates were highly stable in mouse and human plasma. Determination of FR binding affinities using ³H-folic acid and FR⁺ KB tumor cells revealed affinities in the nanomolar range for 2 and 4. The cell uptake of [¹²⁵I]-2 and [^125/131I]-4 proved to be FR specific as it was blocked by the coincubation of folic acid. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in vitro assays were employed for the determination of tumor cell viability upon exposure to [¹³¹I]-4. Compared to untreated control cells, significantly reduced cell viability was observed for FR⁺ cancer cells (KB, IGROV-1, SKOV-3), while FR^- cells (PC-3) were not affected. Biodistribution studies performed in tumor bearing nude mice showed the specific accumulation of both radiofolates in KB tumor xenografts ([¹²⁵I]-2: 3.43 ± 0.28% ID/g; [¹²⁵I]-4: 2.28 ± 0.46% ID/g, 4 h p.i.) and increasing tumor-to-kidney ratios over time. The further improvement of the tumor-to-background contrast was achieved by preinjection of the mice with pemetrexed allowing excellent imaging via single-photon emission computed tomography (SPECT/CT). These findings confirmed the hypothesis that the application of radioiodinated folate conjugates may be a valuable concept to improve tumor-to-background contrast. The inhibitory effect of [ ¹³¹I]-4 on FR⁺ cancer cells in vitro indicates the potential of this class of radiofolates for therapeutic application.

Full text of DOI:10.1021/mp200511t

Article
pubs.acs.org/molecularpharmaceutics
Radioiodinated Folic Acid Conjugates: Evaluation of a Valuable
Concept To Improve Tumor-to-Background Contrast
Josefine Reber,† Harriet Struthers,‡ Thomas Betzel,‡ Alexander Hohn,† Roger Schibli,†,‡
and Cristina Muller*,†
̈
†Center for Radiopharmaceutical Sciences ETH-PSI-USZ, Paul Scherrer Institute, Villigen-PSI, Switzerland
‡Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich, Switzerland
S
* Supporting Information
ABSTRACT: Folic acid radioconjugates can be used for targeting folate receptor
positive (FR+) tumors. However, the high renal uptake of radiofolates is a drawback
of this strategy, particularly with respect to a therapeutic application due to the risk
of damage to the kidneys by particle radiation. The goal of this study was to develop
and evaluate radioiodinated folate conjugates as a novel class of folate-based
radiopharmaceuticals potentially suitable for therapeutic application. Two different
folic acid conjugates, tyrosine-folate (1) and tyrosine-click-folate (3), were
synthesized and radioiodinated using the Iodogen method resulting in [125I]-2
and [125/131I]-4. Both radiofolates were highly stable in mouse and human plasma.
3
Determination of FR binding affinities using H-folic acid and FR+ KB tumor cells
revealed affinities in the nanomolar range for 2 and 4. The cell uptake of [125I]-2
and [125/131I]-4 proved to be FR specific as it was blocked by the coincubation of
folic acid. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in
vitro assays were employed for the determination of tumor cell viability upon
exposure to [131I]-4. Compared to untreated control cells, significantly reduced cell
viability was observed for FR+ cancer cells (KB, IGROV-1, SKOV-3), while FR−
cells (PC-3) were not affected. Biodistribution studies performed in tumor bearing
nude mice showed the specific accumulation of both radiofolates in KB tumor xenografts ([125I]-2: 3.43 0.28% ID/g; [125I]-4:
2.28
0.46% ID/g, 4 h p.i.) and increasing tumor-to-kidney ratios over time. The further improvement of the tumor-to-
background contrast was achieved by preinjection of the mice with pemetrexed allowing excellent imaging via single-photon
emission computed tomography (SPECT/CT). These findings confirmed the hypothesis that the application of radioiodinated
folate conjugates may be a valuable concept to improve tumor-to-background contrast. The inhibitory effect of [131I]-4 on FR+
cancer cells in vitro indicates the potential of this class of radiofolates for therapeutic application.
KEYWORDS: folate receptor, tyrosine-folate, iodine, SPECT/CT, radionuclide therapy
previously investigated the application of radiofolates in
combination with the chemotherapeutic antifolate pemetrexed
(PMX), which was able to improve the tumor-to-kidney ratios
significantly.12−14 The application of PMX in combination with
therapeutic radiofolates would be particularly beneficial if it had
an additional or synergistic tumor growth inhibitory effect.
Radioiodination of tyrosine residues is a common strategy for
efficient radiolabeling of proteins and peptides, demonstrated in
numerous studies of small and large biomolecules. The
instability of radioiodinated tyrosine constructs as a conse-
quence of in vivo deiodination is generally regarded as a
disadvantage for radiopharmaceutical applications. However, in
a recent study, Zardi and co-workers showed that the tumor-to-
INTRODUCTION
■
The folate receptor (FR) emerged as a valuable tumor marker
because of its overexpression in a variety of cancer types and
the fact that it can be targeted by the vitamin folic acid.1−4
Folate-based radiopharmaceuticals have attracted significant
interest for nuclear imaging of cancer with single-photon
emission computed tomography (SPECT; 99mTc, 111In, 67Ga)
and positron emission tomography (PET; 68Ga, 18F) (reviewed
in refs 5−7). However, the FR is also expressed on the luminal
side of proximal tubule cells in the kidneys and thus accessible
to filtered (radio)folates.8−10 The result is a high kidney
accumulation of folate radiopharmaceuticals and hence low
tumor-to-kidney ratios (∼0.1).11 Therefore, there is an inherent
risk of damage to the kidneys by application of therapeutic
radiofolates. Nevertheless, this treatment strategy is attractive in
view of the large number of patients who could benefit from it.
Thus, establishing effective strategies to improve the tumor-to-
kidney ratio of radiofolates is of paramount interest. We have
Received: October 8, 2011
Revised: February 12, 2012
Accepted: April 5, 2012
Published: April 17, 2012
© 2012 American Chemical Society
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kidney ratios of the radioiodinated antibody fragment L19 was
significantly increased over time (∼0.36, 1 h p.i.; ∼ 6.71, 24 h
p.i.).15 In contrast, the same antibody fragment radiolabeled
with 99mTc showed a very low tumor-to-kidney ratio of ∼0.1 at
all time points after injection.16 Thus, we hypothesized that
radioiodinated folate conjugates would exhibit favorable
properties, similar to those found with the radioiodinated
antibody fragment L19. The partial deiodination of radio-
iodinated folate conjugates might result in a quick clearance of
released iodide via kidneys and thus lead to improved tumor-to-
kidney ratios. The design of radioiodinated folic acid conjugates
as an alternative to folate-linked chelates for stable coordination
of radiometals could be an effective strategy to achieve an
improved tumor-to-background contrast.
Another advantage of radioiodinated folate conjugates is the
opportunity to employ a range of iodine radioisotopes suitable
for imaging and therapeutic purposes. The long-lived radio-
isotope iodine-125 (T1/2 = 59.4 d, Eγ = 35.5 keV (6.7%), Auger-
e−) is usually favored for preclinical in vitro and in vivo studies
including small-animal imaging. For clinical applications, the
radiolabeling of biomolecules with iodine-123 (T1/2 = 13.2 h, Eγ
+
+
= 159 keV (83%)) or iodine-124 (T1/2 = 4.18 d, Iβ = 23%, Eβ
(max) = 2140 keV) is useful for SPECT and PET imaging and
can be regarded as diagnostic and dosimetric substitutes for the
−
Figure 1. Chemical structures of folate conjugates 1−4.
therapeutic counterpart iodine-131 (T1/2 = 8.03 d, Iβ = 100%,
−
Eβ (average) = 182 keV; Eγ = 365 keV (82%)).
performed at room temperature for 5 min with [131I]iodine
or 15 min with [125I]iodine. Quality control was performed by
high-performance liquid chromatography (HPLC) as reported
in detail in the SI (section 1, Figure S3). The radioiodinated
compounds [125I]-2 (retention time (Rt) ≈ 14.4 min) and
The goal of this study was to evaluate the proposed concept
of partial deiodination of radioiodinated folate conjugates as a
method to improve the tumor-to-kidney ratio of radiofolates.
We therefore prepared two structurally different folate
conjugates, the tyrosine-folate (1) and the tyrosine-click-folate
(3). The 125I-radioiodinated folate tracers [125I]-2 and [125I]-4
were evaluated in vitro using FR positive KB tumor cells. To
assess the tissue distribution of radioactivity, animal experi-
ments including SPECT/CT imaging studies were performed.
The novel radiofolates were also tested in combination with
PMX. The second objective of this study was to investigate the
cytotoxic effect of the 131I-radioiodinated tyrosine-click-folate
([131I]-4) in vitro by employing a standard cell viability assay.
By application of FR targeted in vitro therapy using the particle-
emitting radioiodine-131 in combination with PMX, a
potentially enhanced effect of these two treatment modalities
was assessed using FR positive and FR negative cancer cell
lines.
[
125/131I]-4 (Rt ≈ 15.7 min) were separated from traces of free
iodide (Rt ≈ 2.9 min) and cold precursor 1 (Rt ≈ 12.4 min) or
3 (Rt ≈ 13.9 min) by means of HPLC using the same method
as established for analysis. For in vitro and in vivo experiments
methanol in the collected HPLC fraction was evaporated, and
the radiofolate solution was diluted with phosphate-buffered
saline (PBS) at pH 7.4. The determination of the octanol/PBS
distribution coefficient (log D value) is reported in the SI
(section 2).
Stability Experiments. The stabilities of [125I]-2 and
[
125I]-4 were determined in mouse and human plasma. HPLC-
purified radiofolates (∼2.0 MBq, 50 μL) were mixed with
250 μL of mouse or human plasma and incubated at 37 °C.
Aliquots were taken for analysis at defined time points after
incubation (0, 4, 24, 48, 72, and 168 h). Plasma samples of 50
μL were precipitated with 200 μL of methanol and centrifuged
twice before injection into the HPLC. In vitro studies for the
determination of the metabolic stability were performed using
murine liver microsomes and are reported in the SI (section 3,
Figure S4). In vivo studies in mice performed to determine
potential metabolites and/or deiodination are reported in the
SI (section 4, Table S2).
Cell Culture. KB cells (human cervix carcinoma cell line,
ACC-136) and PC-3 (human prostate carcinoma cell line,
ACC-465) were purchased from the German Collection of
Microorganisms and Cell Cultures (DSMZ, Braunschweig,
Germany). IGROV-1 cells (human ovarian carcinoma cell line)
were a kind gift from Dr. Gerrit Jansen (Department of
Rheumatology, VU University Medical Center, Amesterdam,
The Netherlands). SKOV-3 i.p. cells (human ovarian
adenocarcinoma cancer cell line, herein referred as to
SKOV-318) were kindly provided by Dr. Ilse Novak-Hofer
EXPERIMENTAL SECTION
■
Radioiodination. The synthesis of compounds 1 to 4
(Figure 1) is reported in the Supporting Information (SI,
section 1, Figures S1−S3, Table S1). Compounds 1 and 3 were
radiolabeled with [125I]iodine and [131I]iodine isotopes
obtained from Perkin-Elmer (Waltham, MA, USA). Specific
activities were indicated as ∼629 GBq/mg (∼ 78.6 MBq/nmol)
for [125I]iodine and >185 GBq/mg (>24.2 MBq/nmol) for
[
131I]iodine. Radioiodination was performed using the Iodogen
method as previously described.17 In brief, sodium radioiodide
125I: up to 60 MBq; 131I: up to 45 MBq) dissolved in
(
tris(hydroxymethyl)aminomethane (TRIS) buffer pH 7.6 was
added to an Iodogen-coated (0.05 μg) reaction vial. The
oxidation reaction was allowed to proceed for 5 min at room
temperature. The solution containing the activated 125I or 131I
was removed from the insoluble oxidizing agent and transferred
to a reaction vial containing 10−30 μL of the folate precursor 1
or 3 (1 mM), respectively. The labeling procedure was
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(Center for Radiopharmaceutical Sciences ETH-PSI-USZ, Paul
Scherrer Institute, Switzerland). KB cells are known to express
the FR at very high levels, whereas IGORV-1 and SKOV-3 cells
express the FR at lower levels.1,19,20 PC-3 cells that do not
express the FR were used as a negative control.21 The cells were
cultured as monolayers at 37 °C in a humidified atmosphere
containing 5% CO2. PC-3 cells were cultured in Roswell Park
Memorial Institute (RPMI) 1640 medium (Amimed, Bio-
concept, Switzerland). Importantly, KB, IGROV-1, and
SKOV-3 cells were cultured in a folate free RPMI cell culture
medium referred to as FFRPMI (without folic acid, vitamin B12,
and phenol red; Cell Culture Technologies GmbH, Gravesano/
Lugano, Switzerland). FFRPMI and RPMI media were
supplemented with 10% heat-inactivated fetal calf serum, L-
glutamine, and antibiotics. Routine culture treatment was
performed twice a week.
Cell Uptake and Internalization. KB cells were seeded in
12-well plates to grow overnight (∼700 000 cells in 2 mL
FFRPMI medium/well). HPLC purified compounds [125I]-2
and [125I]-4 (∼0.04 MBq, ∼0.5 pmol) were added to each well.
In some cases cells were incubated with excess folic acid to
block FRs on the surface of KB cells. After incubation at 37 °C
for different time periods (5, 15, 30, 60, 120, and 240 min), the
cells were washed three times with PBS to determine total
radiofolate uptake. To assess the internalized fraction of
radiofolate, KB cells were additionally washed with a stripping
buffer (aqueous solution of 0.1 M acetic acid and 0.15 M NaCl,
pH 3)22 to release FR bound radiofolates from the cell surface.3
Cell lysis was accomplished by the addition of 1 mL of 1 N
NaOH to each well. The cell suspensions were transferred to
4 mL tubes, and each sample was counted in a γ-counter. After
homogenization by vortex, the protein concentration was
determined for each sample using a Micro BCA Protein Assay
kit. The measured radioactivity was standardized to the average
content of 0.3 mg of protein in a single well.
medium was removed, and the cells were incubated at 37 °C
with 200 μL of FFRPMI medium (without supplements)
containing [131I]-4 (0.50 MBq/mL, corresponding to 20 nM
folate tracer) alone or in combination with excess folic acid
(200 nM) to block FRs. To determine the amount of
radiofolate [131I]-4 necessary to reduce cell viability to 50%
of untreated cells, KB, IGROV-1 and SKOV-3 cells were
incubated for 4 h at 37 °C with 200 μL of FFRPMI medium
(without supplements) containing [131I]-4 (10.0, 5.0, 1.0, 0.5,
0.1, 0.05, and 0.01 MBq/mL). For the investigation of a
potential effect of PMX (Alimta, LY231514; Eli Lilly, Bad
Homburg, Germany) on cell viability, KB cells were incubated
with [131I]-4 (0.01 MBq/mL) or PMX (1 μM) alone or with a
combination of these two agents for 4 or 24 h at 37 °C. Cell
incubation with FFRPMI medium only was performed as a
control experiment. After incubation, the cells were washed
with 200 μL of PBS, and 200 μL of supplemented FFRPMI or
RPMI medium was added. Four days later, 30 μL of a filtered
MTT solution in PBS (5 mg/mL) was added to each well, and
incubation was continued for an additional 4 h at 37 °C. The
medium was removed, and the dark-violet formazan crystals
were dissolved in 200 μL of dimethyl sulfoxide (DMSO). The
absorbance was determined at 560 nm using a microplate
reader (Victor X3, Perkin-Elmer). To quantify cell viability, the
ratio of the absorbance of the samples to the absorbance of
control cell samples (= 100% viability) was calculated.
Biodistribution Studies. In vivo experiments were
approved by the local veterinarian department and conducted
in accordance with the Swiss law of animal protection. Six- to
eight-week-old female, athymic nude mice (CD-1 Foxn-1/nu)
were purchased from Charles River Laboratories (Sulzfeld,
Germany). The animals were fed with a folate deficient rodent
diet (Harlan Laboratories, Indianapolis, IN, U.S.) starting 5
days prior to tumor cell inoculation.23 Mice were inoculated
with KB cells (5 × 106 cells in 100 μL of PBS) into the subcutis
of each shoulder. Animal experiments were performed
approximately 14 days after tumor cell inoculation. Biodis-
tribution studies were performed in triplicate. Radiofolates
In Vitro Binding Affinity Assays. Binding assays with
nonradioactive reference compounds 2 and 4 were performed
with KB tumor cells suspended in PBS at pH 7.4 (7000 cells/
240 μL per Eppendorf tube). The cells were incubated in
[
125I]-2 and [125I]-4 were diluted in PBS pH 7.4 for immediate
3
triplicate with H-folic acid (10 μL, 0.84 nM) and increasing
administration via a lateral tail vein (0.2 MBq, 2.5 pmol, 100 μL
per mouse). Potassium iodide was dissolved in PBS and
injected intraperitoneally (4 mg, 200 μL), 1 h prior to the
radioactivity. Blocking experiments were performed 4 h after
injection, with mice coinjected with excess folic acid dissolved
in PBS (100 μg, 100 μL). The animals were sacrificed at 1, 4, or
24 h after administration of the radiofolate. Selected tissues and
organs were collected, weighed, and counted for radioactivity in
a γ-counter. The results were recorded as the percentage of the
injected dose per gram of tissue weight [% ID/g].
Biodistribution studies performed with [125I]-4 in combination
with preinjected PMX are reported in the SI (section 5, Figure
S5).
concentrations of the nonradioactive reference compound (5.0
× 10−7 to 5.0 × 10−12 M in 250 μL of PBS at pH 7.4) on a
shaker at 4 °C for 30 min. Nonspecific binding was determined
in the presence of excess folic acid (10−3 M). After incubation,
Eppendorf tubes containing the cell suspensions were
centrifuged at 4 °C for 5 min, and the supernatant was
removed. By addition of 0.5 mL of NaOH (1 N) the cell pellets
were lysed and transferred into scintillation tubes containing
5 mL of scintillation cocktail (Ultima Gold; Perkin-Elmer). The
radioactivity was measured using a liquid scintillation analyzer
(Tri-Carb 1900 TR, Packard), and an inhibitory concentration
of 50% was determined from displacement curves using
GraphPad Prism (version 5.01) software. Relative affinities
were defined as the inverse molar ratio of test compounds
Statistical analyses were performed by using a t-test
(Microsoft Excel software). All analyses were two-tailed and
considered as type 3 (two sample unequal variance). A p-value
of <0.05 was considered statistically significant.
3
required to displace 50% of H-folic acid, and the relative FR
affinity of folic acid was set to 1.
MTT Assay. The inhibition of cell growth was evaluated
using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assays. KB, IGROV-1, SKOV-3, and PC-3
cells were harvested and seeded in 96-well plates at 2.5 × 103
cells per well in a final volume of 200 μL of FFRPMI medium
(KB, IGROV-1, and SKOV-3) or RPMI medium (PC-3) with
supplements. After 24 h incubation for cell adhesion, the
SPECT/CT Imaging Studies and ex Vivo Autoradiog-
raphy. Imaging experiments were performed using an X-
SPECT system (Gamma Medica Inc., Northridge, CA, USA)
with a small animal single-head SPECT/CT device. Radiofolate
[
125I]-4 (∼5.0 MBq; ∼64 pmol) was injected into a lateral tail
vein of KB tumor-bearing nude mice. PMX was diluted with
NaCl to 0.9% according to the instructions of the manufacturer
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and injected into a lateral tail vein (400 μg, 100 μL), 1 h before
the radiotracer.12,13 SPECT scans of 60 min duration were
performed approximately 24 h after injection of the radiotracer
followed by CTs. SPECT data were collected and reconstructed
using the software LumaGEM (version 5.407; Gamma Medica
Inc.). CT data were acquired with an X-ray CT system
(Gamma Medica Inc.) and reconstructed with the software
COBRA (version 4.5.1). The fusion of SPECT and CT data was
performed with IDL Virtual Machine (version 6.0) software.
Images were generated by the software Amira (version 4.0.1).
Ex vivo autoradiography studies are reported in the SI (section
6).
and values higher than 1.0 reflect a stronger affinity.25,26 For
our test compounds the determination of binding affinities
resulted in relative values of 0.78 for I-tyrosine-folate (2) and
0.67 for I-tyrosine-click-folate (4).
In vitro stability experiments were performed in both mouse
and human plasma. Within the time period of investigation
(7 days), HPLC analysis of the collected and precipitated
plasma samples showed always only one single peak with an
integrated area of 100% and a retention time that corresponded
to the radioiodinated folate conjugates ([125I]-2 or [125I]-4).
Thus, no signs of deiodination or decomposition of the
radioiodinated compounds [125I]-2 and [125I]-4 was deter-
mined. In addition, potential deiodination of [125I]-2 or [125I]-
4) was investigated using murine liver microsomes. However, in
contrast to [125I]iodo-L-tyrosine which was almost completely
deiodinated within 30 min, the deiodination of the radiofolates
([125I]-2 or [125I]-4) was not determined over a time period of
24 h (SI, section 3, Figure S4).
In Vitro Cell Viability Study. An MTT assay was employed
to test the effect of the 131I-radiolabled folate conjugate ([131I]-
4, Figure 3). In the absence of folic acid, the incubation of FR-
positive cells with 131I-tyrosine-click-folate ([131I]-4, 5 MBq/
mL) reduced viability of KB, IGROV-1, and SKOV-3 cells to
4.5%, 15.3%, and 27.4% of untreated control cells (Figure 3A).
However, if the cells were coincubated with excess folic acid to
block FRs, cell viability was not affected by application of
radiofolate [131I]-4 at the same radioactivity concentrations. In
addition, no inhibition of cell viability was observed with the
same amount of [131I]-4 in control experiments performed with
FR negative PC-3 cells (Figure 3A). The determination of the
amount of [131I]-4 necessary to reduce cell viability of KB,
IGROV-1, and SKOV-3 cells to 50% of untreated control cells
revealed radioactivity concentrations of 0.39 0.03 MBq/mL,
0.95 0.05 MBq/mL, and 1.22 0.09 MBq/mL, respectively
(Figure 3B).
RESULTS
■
Synthesis and Radioiodination. Compounds 1−4 are
shown in Figure 1. The organic synthesis and chemical
characterization are reported in the SI (Figures S1 and S2,
Table S1). Radioiodination of folate compounds 1 and 3 was
obtained with 125/131I, in a radiochemical yield of >97%. The
shift to longer HPLC retention times of radiolabeled
compounds [125I]-2 and [125/131I]-4 enabled their separation
from the unlabeled precursors 1 and 3 to obtain the highest
possible specific activities corresponding to the specific
activities of the radionuclides (∼629 GBq/mg, i.e., ∼78.6
MBq/nmol for 125I and >185 GBq/mg, i.e., >24.2 MBq/nmol
for 131I; SI, Figure S3).
In Vitro Evaluation. Cell uptake studies showed 125I-
tyrosine-folate ([125I]-2) and 125I-tyrosine-click-folate ([125I]-4)
had similar characteristics and were comparable to previously
evaluated radiofolates.24 The maximal amount of uptake was
reached after an incubation time of about 90−120 min at 37
°C. The internalized fraction of both radiofolates accounted for
25−30% of total cell uptake (sum of FR bound radiofolate on
the cell surface and internalized fraction). Co-incubation of
excess folic acid resulted in an inhibition of radiofolate uptake
for both [125I]-2 and [125I]-4 to less than 0.1% of the total
added radioactivity (Figure 2).
The incubation of KB cells with PMX (1 μM) for 4 h and 24
h, respectively, reduced tumor cell viability to 80% and 50% of
untreated controls (Figure 4). The application of the [131I]-4 at
a concentration of 0.01 MBq/mL did not affect tumor cell
viability if applied as a single agent for 4 h. However, if this
radioactivity concentration was applied in combination with
PMX (1 μM), it resulted in an enhanced inhibitory effect,
decreasing cell viability to 72% (after 4 h) and 23% (after 24 h)
compared with untreated controls.
Relative binding affinities were determined using a
3
competition assay with H-folic acid and the nonradioactive
reference compounds 2 and 4. The binding affinity of folic acid
was set to 1.0. A relative affinity value of 1.0 implies that the
test compound has an affinity for the FR equal to that of folic
acid. Likewise, values lower than 1.0 reflect a weaker affinity,
Biodistribution Studies. Biodistribution data for [125I]-2
and [125I]-4 are shown in Table 1. Both radiotracers
accumulated in FR positive KB tumor xenografts and in the
kidneys. The tumor uptake of 125I-tyrosine-folate ([125I]-2: 4.42
0.66% ID/g) was higher than the uptake of 125I-tyrosine-
click-folate ([125I]-4: 2.36 0.05% ID/g) at 1 h after injection
(p = 0.02). However, tumor retention over time was superior
for [125I]-4 (1.92 0.24% ID/g, 24 h p.i.) compared to [125I]-2
(1.25 0.18% ID/g, 24 h p.i.). The reduction of the tumor
uptake after coinjection of excess folic acid (4 h p.i.) was
pronounced in the case of [125I]-4 (<2% of control values),
whereas in the case of [125I]-2 tumor uptake could only be
reduced to 48% of the control values.
The renal accumulation of radioactivity was high (∼19% ID/
g) 1 h after injection for both radiofolates. However, 4 h after
injection there was a significant difference (p = 0.03) between
Figure 2. Cell binding (A) and internalization (B) of 125I-labeled
tyrosine-folate ([125I]-2) and tyrosine-click-folate ([125I]-4) in KB cells
after an incubation time of 120 min at 37 °C. Blockade (C) of FR by
coincubation with excess folic acid resulted in an uptake of less than
1%.
[
125I]-2 (9.80 1.67% ID/g) and [125I]-4 (16.44 2.64% ID/
g). At 24 h after injection this difference was even more
pronounced ([125I]-2: 0.85 0.06% ID/g vs [125I]-4: 7.10
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Figure 3. (A) Effects of 131I-tyrosine-click-folate [131I]-4 (5 MBq/mL, corresponding to 200 nM folate conjugate) on FR-positive KB, IGROV-1, and
SKOV-3 cells and FR-negative PC-3 cells after incubation for 4 h at 37 °C in the presence and absence of excess folic acid (FA). (B) Determination
of the concentration of [131I]-4 (MBq/mL) which is necessary to reduce cell viability to 50% of untreated control cells (KB: 0.39 0.03 MBq/mL;
IGROV-1: 0.95 0.05 MBq/mL; and SKOV-3: 1.22 0.09 MBq/mL).
(Figure 5B). SPECT/CT scans of a mouse that received both
potassium iodide and PMX prior to the injection of [125I]-4
resulted in radioactivity uptake that was restricted to FR
positive tumor xenografts, and only traces of radioactivity were
observed in nontargeted organs and tissues (Figure 5C).
Images of ex vivo autoradiography studies are shown in
Figure 6. The quantification of radioactivity uptake in tumor
and kidney sections of the mouse that received PMX showed
an uptake reduced to 56% (tumor) and 4% (kidney) of controls
(SI, section 6).
DISCUSSION
■
Figure 4. Percentage of viable KB cells after incubation for 4 and 24 h
at 37 °C (A) with culture medium only (= 100%, control experiment),
(B) with PMX (1 μM), (C) with 131I-tyrosine-click-folate ([131I]-4:
0.01 MBq/mL), and (D) with the combination of PMX and [131I]-4.
Two different folic acid conjugates were synthesized and
evaluated. In compound 1 tyrosine was conjugated to the γ-
carboxyl group of the glutamate entity of folic acid, whereas in
compound 3 tyrosine was conjugated to an azide-derivatized
folic acid27 via a “click” reaction, which resulted in a triazole-
containing linker entity. Both folate conjugates (1 and 3) were
readily radioiodinated using the Iodogen method. Separation
from cold precursors gave high specific activities of [125I]-2 and
1.44% ID/g; p = 0.02). The application of [125I]-4 after
preinjection of PMX resulted in a significantly reduced uptake
of radioactivity in the kidneys, while uptake in the tumor
xenografts was largely retained (Table 2 and SI, section 5, S5).
Radioactivity in the blood circulation was significantly higher
for [125I]-2 compared to [125I]-4 (1 h p.i.; p = 0.01). As a
consequence, accumulation in nontargeted organs and tissues
was clearly higher after administration of radiofolate [125I]-2
compared to [125I]-4. The liver accumulation of radioactivity
after injection of [125I]-2 was high (>16% ID/g, 1 h p.i. and
>2% ID/g, 4 h p.i.), but almost negligible in the case of [125I]-4
(<0.3% ID/g, 1 h p.i., p < 0.01, and <0.2% ID/g, 4 h p.i., p =
0.01). On the other hand, uptake in the gastrointestinal tract
was comparable for both radiotracers (∼4.5% ID/g, 1 h p.i.).
With regard to uptake in the thyroid glands, large amounts of
radioactivity (>1000% ID/g, 4 h p.i.) were found for [125I]-2,
whereas a more than 10-fold lower accumulation was observed
in the case of [125I]-4. Intraperitoneal administration of
potassium iodide 1 h prior to administration of the radiotracer
reduced the undesired uptake of iodide in the thyroid glands
significantly ([125I]-2: 13.74 5.01% ID/g and [125I]-4: 2.53
0.39% ID/g, 4 h p.i.).
[
125/131I]-4. The in vitro stability in human and murine plasma
was high for both radiofolates. FR binding affinity and cell
uptake were almost identical for both compounds and
comparable to previously evaluated radiofolates.24,28 In vivo,
the 125I-tyrosine-folate ([125I]-2) was significantly instable. As a
consequence, the high uptake of radioactivity was found in
nontarget tissues and organs at early time points after injection
of [125I]-2. An increasingly high uptake of radioactivity was
found in the thyroid gland which could be ascribed to the
accumulation of free [125I]-iodide. However, a significant
reduction of radioactivity in the thyroid gland was achieved
in mice that received a preinjection of potassium iodide. The
low stability of [125I]-2 was further proven by the detection of
significant amounts of free [125I]-iodide in urine and blood
samples (SI, Table S2). In contrast, free [125I]-iodide was not
detectable in blood samples after the injection of [125I]-4, and
only a low accumulation of radioactivity was found in the
thyroid gland. The increased in vivo stability of [125I]-4
compared to [125I]-2 could be due to the “click” conjugation
of the tyrosine entity, which may make the compound more
resistant toward enzymatic attack.
SPECT/CT Imaging and Ex Vivo Autoradiography.
Figure 5 shows SPECT/CT images of mice after administration
of the radiotracer [125I]-4. The administration of potassium
iodide effectively blocked the uptake of radioactivity in the
thyroid gland (Figure 5A). After injection of PMX, renal
retention of radioactivity was reduced to background levels
Tumor uptake of the 125I-tyrosine-folate ([125I]-2) was
significantly higher than for the 125I-tyrosine-click-folate
([125I]-4) at 1 and 4 h p.i. In the case of [125I]-2 it was not
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Table 1. Biodistribution Data of [125I]-2 and [125I]-4 in KB Tumor-Bearing Nude Mice
a
125I-tyrosine-folate ([125I]-2), mean SD [%ID/g]
b
c
dissection time
1 h p.i.
4 h p.i.
24 h p.i.
folic acid, 4 h p.i.
KI, 4 h p.i.
blood
3.00 0.56
2.66 0.42
2.25 0.58
18.33 2.47
9.44 3.19
4.30 1.01
16.53 1.20
13.32 2.92
261 67
2.15 0.42
1.72 0.25
1.60 0.26
9.80 1.67
7.18 0.62
1.65 0.90
2.74 0.45
16.53 3.42
1059 184
0.82 0.30
1.01 0.24
3.43 0.28
1.65 0.41
1.28 0.23
0.36 0.06
0.09 0.02
0.11 0.02
0.08 0.03
0.85 0.06
0.20 0.04
0.07 0.01
0.09 0.01
1.36 0.11
2830 1272
0.02 0.00
0.03 0.01
1.25 0.18
14.38 3.66
14.45 1.88
1.47 0.14
2.25 1.00
1.74 0.68
1.72 0.93
1.79 0.58
9.08 5.49
2.25 1.43
0.83 0.32
15.34 9.09
868 367
0.55 0.24
1.01 0.53
1.65 0.61
0.74 0.04
1.98 0.18
0.90 0.09
2.71 0.52
2.04 0.41
1.26 0.15
8.66 1.86
4.44 1.52
1.68 0.48
2.90 0.63
4.02 0.66
13.74 5.01
0.53 0.10
0.98 0.20
4.03 0.22
1.53 0.30
1.43 0.24
0.48 0.09
lung
spleen
kidneys
stomach
intestines
liver
salivary glands
thyroid gland
muscle
1.45 0.26
1.59 0.28
4.42 0.66
1.48 0.18
0.27 0.04
0.24 0.03
bone
tumor
tumor-to-blood
tumor-to-liver
tumor-to-kidney
a
125I-tyrosine-click-folate ([125I]-4), mean SD [%ID/g]
b
c
dissection time
control, 1 h p.i.
control, 4 h p.i.
control, 24 h p.i.
folic acid, 4 h p.i.
KI, 4 h p.i.
blood
0.10 0.01
0.59 0.14
0.20 0.02
19.12 0.58
0.94 0.29
4.53 1.77
0.28 0.08
6.11 1.37
8.36 1.77
0.71 0.13
0.55 0.05
2.36 0.05
23.21 2.17
8.73 2.13
0.12 0.00
0.18 0.15
0.31 0.09
0.14 0.05
16.44 2.64
0.35 0.08
1.24 0.01
0.15 0.03
4.01 1.31
10.81 4.99
0.32 0.07
0.23 0.05
2.28 0.46
17.41 9.66
15.19 3.99
0.14 0.02
0.01 0.00
0.05 0.03
0.02 0.00
7.10 1.44
0.12 0.10
0.10 0.01
0.02 0.00
0.42 0.22
47.59 28.07
0.04 0.04
0.02 0.01
1.92 0.24
286.75 53.44
84.82 12.71
0.27 0.05
0.03 0.00
0.03 0.00
0.02 0.01
0.08 0.00
0.26 0.31
0.46 0.30
0.02 0.00
0.10 0.04
9.53 8.10
0.01 0.00
0.02 0.00
0.03 0.01
1.08 0.47
2.20 0.96
0.41 0.15
0.08 0.01
0.23 0.03
0.14 0.02
17.84 2.60
1.01 1.00
2.84 1.16
0.16 0.03
3.49 0.98
2.47 0.56
0.40 0.04
0.30 0.01
2.53 0.39
30.93 6.87
15.78 4.33
0.14 0.02
lung
spleen
kidneys
stomach
intestines
liver
salivary glands
thyroid gland
muscle
bone
tumor
tumor-to-blood
tumor-to-liver
tumor-to-kidney
a
b
c
∼2.5 pmol/mouse. FR blockade with folic acid (100 μg) i.v. coinjected. Thyroid blockade with KI (4 mg) i.p. injected 1 h before the radiofolate.
Table 2. Biodistribution Data 4 h p.i. of [125I]-4 in Combination with PMX in KB Tumor-Bearing Nude Mice
a
125I-tyrosine-click-folate ([125I]-4), mean SD [%ID/g]
b
b
b
control
PMX, 400 μg
PMX, 200 μg
PMX, 100 μg
tumor
2.28 0.46
0.18 0.15
0.15 0.03
16.44 2.64
17.41 9.66
15.19 3.99
0.14 0.02
1.55 0.17
0.50 0.75
0.88 1.38
1.08 0.06
16.06 11.85
13.50 10.38
1.44 0.21
1.97 0.26
0.07 0.01
0.09 0.01
1.36 0.26
29.45 6.14
22.37 4.27
1.49 0.35
2.97 0.60
0.17 0.12
0.18 0.03
5.17 3.00
23.92 12.54
16.43 2.69
0.72 0.40
blood
liver
kidney
tumor-to-blood
tumor-to-liver
tumor-to-kidney
a
b
∼2.5 pmol/mouse. PMX was injected 1 h before the radiofolate.
possible to block the tumor uptake to background levels with
excess folic acid. This indicates unspecific accumulation of
radioactivity in the tumor, which can be ascribed to free [125I]-
iodide circulating in the blood. A relatively high accumulation
of radioactivity was also found in the liver shortly after injection
of [125I]-2 (>16% ID/g, 1 h p.i.) which might also be due to
significant amounts of free [125I]-iodide in the blood circulation.
On the other hand, liver uptake of [125I]-4 was very low (<1%
ID/g) compared to previously evaluated radiofolates.28,29
Unfavorable accumulation of radioactivity in the intestinal
tract early after injection was found with both radiofolates
([125I]-2 and [125I]-4). This was possibly a result of their more
lipophilic character compared to folate-based radiometal
complexes (e.g. 111In-DOTA-folate) as shown by determination
of log D values ([125I]-2: −2.87 0.02 and [125I]-4: −3.13
0.07; SI).
At early time points after injection (1 h p.i.), the tumor-to-
kidney ratios of radioactivity were low ([125I]-2: 0.24 0.03;
[
125I]-4: 0.12
0.00).29 However, over time the tumor-to-
kidney ratios increased ([125I]-2: 1.47 014; [125I]-4: 0.27
0.05; 24 h p.i.), which confirmed our hypothesis that partial
deiodination may improve the tissue distribution. In addition,
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kidney ratios (4 h p.i.: ∼1.5 and 24 h p.i.: ∼3.0) of [125I]-4
(Table 2 and SI, section 5, Figure S4). These findings could be
confirmed by ex vivo autoradiography (SI) and SPECT/CT
imaging studies. However, it has to be critically acknowledged
that the images were taken the day after injection of the
radiofolate when unspecifically accumulated radiofolate had
been excreted from the intestinal tract.
We were able to demonstrate in vitro that [131I]-4 reduced
the viability of KB, IGROV-1, and SKOV-3 cells, but that the
effect was not observed if FRs were blocked by incubation of
these cells with excess folic acid. In addition, no inhibition of
viability was observed in FR-negative PC-3 cells, which proved
FR-mediated cytotoxicity. The determination of the radio-
activity concentration of [131I]-4 necessary to reduce cell
viability to 50% of untreated control cells showed that KB cells
were more sensitive to radiofolate treatment than IGROV-1
and SKOV-3 cells. These findings are in agreement with the
lower FR-expression levels on IGROV-1 and SKOV-3 cells
compared to KB cells which express FRs at very high
levels.1,19,20 Recently, Bischof et al. demonstrated that PMX
displayed radiosensitizing effects in combination with external
radiation in vitro.31,32 In the present study it could be shown
that the application of PMX in combination with [131I]-4
enhanced the inhibitory effect on FR positive tumor cell
viability. It is likely that the coapplication of therapeutic
radiofolates and PMX could also enhance FR targeted
radionuclide therapy in vivo similar to the application of 5-
fluorouracil, which has been applied in the clinics in
combination with peptide and antibody-based targeted radio-
nuclide therapies.33−35
In this study we were able to demonstrate that radioiodinated
folate conjugates would be favorable with respect to a
therapeutic application because partial in vivo deiodination
results in low kidney retention of radioactivity over time. On
the other hand, we also experienced that too low stability as is
the case for [125I]-2 would be less advantageous as it results in
unspecific uptake of radioactivity in nontargeted tissues. With
respect to the improved tracer design, finding an optimal
balance between sufficient plasma stability and partial in vivo
deiodination of the radiofolate would be desirable. Never-
theless, we believe that radioiodinated folate conjugates provide
a suitable concept for FR targeted radionuclide therapy,
particularly in combination with PMX, which we proved for
the first time results in an enhanced anticancer effect in vitro.
Figure 5. SPECT/CT images of KB tumor-bearing mice 24 h after
injection of 125I-tyrosine-click-folate ([125I]-4): (A) with preinjected
KI, (B) with preinjected PMX, and (C) with preinjected KI and PMX.
(tu = tumor; ki = kidney; thy = thyroid gland; int = intestines; bl =
urinary bladder).
ASSOCIATED CONTENT
■
S
* Supporting Information
Organic syntheses and radioiodination, octanol/PBS distribu-
tion coefficient, investigation of metabolic stability using liver
microsomes, metabolite studies, biodistribution of [125I]-4 in
combination with pemetrexed, and ex vivo autoradiography.
This material is available free of charge via the Internet at
http://pubs.acs.org.
as previously shown with other radiofolates, preinjection of
PMX reduced kidney uptake in a dose-dependent manner
without significantly affecting tumor accumulation.14,30 Thus,
predosing of PMX resulted in unprecedentedly high tumor-to-
AUTHOR INFORMATION
■
Corresponding Author
*Paul Scherrer Institute, Center for Radiopharmaceutical
Sciences ETH-PSI-USZ 5232, Villigen-PSI, Switzerland. E-
mail: cristina.mueller@psi.ch. Phone: +41-56-310 44 54; fax:
+41-56-310 28 49.
Figure 6. Ex vivo autoradiography of tissue sections after the injection
of 125I-tyrosine-click-folate [125I]-4 from KB tumors (A and B) and
kidneys (C and D) from a control mouse (A and C) and a mouse that
received preinjected PMX (B and D).
Notes
The authors declare no competing financial interest.
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mice by anti-L1-cell adhesion molecule monoclonal antibody treat-
ment. Cancer Res. 2006, 66 (2), 936−43.
ACKNOWLEDGMENTS
■
We thank Nadja Romano, Alain Blanc, and Kurt Hauenstein for
technical assistance. This project was supported by the Swiss
National Science Foundation (Ambizione PZ00P3_121772)
and COST (Action BM0607).
(19) Ke, C. Y.; Mathias, C. J.; Green, M. A. The folate receptor as a
molecular target for tumor-selective radionuclide delivery. Nucl. Med.
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(20) Garin-Chesa, P.; Campbell, I.; Saigo, P. E.; Lewis, J. L.; Old, L.
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suicide gene therapy in human prostate cancer and nasopharyngeal
cancer with herpes simplex virus thymidine kinase. Cancer Gene Ther.
2005, 12 (10), 796−809.
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