Alternative chelator for 89Zr radiopharmaceuticals: Radiolabeling and evaluation of 3,4,3-(LI-1,2-HOPO)

Article abstract of DOI:10.1021/jm500389b

Zirconium-89 is an effective radionuclide for antibody-based positron emission tomography (PET) imaging because its physical half-life (78.41 h) matches the biological half-life of IgG antibodies. Desferrioxamine (DFO) is currently the preferred chelator for ⁸⁹Zr⁴⁺; however, accumulation of ⁸⁹Zr in the bones of mice suggests that ⁸⁹Zr⁴⁺ is released from DFO in vivo. An improved chelator for ⁸⁹Zr⁴⁺ could eliminate the release of osteophilic ⁸⁹Zr⁴⁺ and lead to a safer PET tracer with reduced background radiation dose. Herein, we present an octadentate chelator 3,4,3-(LI-1,2-HOPO) (or HOPO) as a potentially superior alternative to DFO. The HOPO ligand formed a 1:1 Zr-HOPO complex that was evaluated experimentally and theoretically. The stability of ⁸⁹Zr-HOPO matched or surpassed that of ⁸⁹Zr-DFO in every experiment. In healthy mice, ⁸⁹Zr-HOPO cleared the body rapidly with no signs of demetalation. Ultimately, HOPO has the potential to replace DFO as the chelator of choice for ⁸⁹Zr-based PET imaging agents.

Full text of DOI:10.1021/jm500389b

Article
pubs.acs.org/jmc
89
Alternative Chelator for Zr Radiopharmaceuticals: Radiolabeling
and Evaluation of 3,4,3-(LI-1,2-HOPO)
Melissa A. Deri,†,‡,§ Shashikanth Ponnala,†,‡ Brian M. Zeglis,† Gabor Pohl,∥ J. J. Dannenberg,*,∥
Jason S. Lewis,*,† and Lynn C. Francesconi*,‡,§
†Department of Radiology and the Program in Molecular Pharmacology and Chemistry, Memorial Sloan-Kettering Cancer Center,
1275 York Avenue, New York, New York 10065, United States
‡Department of Chemistry, Hunter College of the City University of New York, 695 Park Avenue, New York, New York 10065,
United States
§Department of Chemistry, The Graduate School of the City University of New York, 365 Fifth Avenue, New York, New York 10016,
United States
∥Department of Chemistry, City University of New YorkHunter College and the Graduate School, 695 Park Avenue, New York,
New York 10065, United States
S
* Supporting Information
ABSTRACT: Zirconium-89 is an effective radionuclide for
antibody-based positron emission tomography (PET) imaging
because its physical half-life (78.41 h) matches the biological
half-life of IgG antibodies. Desferrioxamine (DFO) is currently
the preferred chelator for 89Zr4+; however, accumulation of
89Zr in the bones of mice suggests that 89Zr4+ is released from
DFO in vivo. An improved chelator for 89Zr4+ could eliminate
the release of osteophilic 89Zr4+ and lead to a safer PET tracer
with reduced background radiation dose. Herein, we present an octadentate chelator 3,4,3-(LI-1,2-HOPO) (or HOPO) as a
potentially superior alternative to DFO. The HOPO ligand formed a 1:1 Zr-HOPO complex that was evaluated experimentally
and theoretically. The stability of 89Zr-HOPO matched or surpassed that of 89Zr-DFO in every experiment. In healthy mice, 89Zr-
HOPO cleared the body rapidly with no signs of demetalation. Ultimately, HOPO has the potential to replace DFO as the
chelator of choice for 89Zr-based PET imaging agents.
89Zr is readily attracting attention as a radionuclide for
INTRODUCTION
■
positron emission tomography (PET) imaging.5−12 In the past
The past few decades have played witness to a revolution in the
several years, a wide variety of preclinical studies have been
understanding of the intersection between transition metals and
medicine. Not only has the body’s management of metals
emerged as an important therapeutic target, but metals have
become critical components of diagnostic and therapeutic
published.13−19 A number of 89Zr-based imaging agents have
been translated into the clinic, including five current clinical
trials in the U.S. alone.20−25 While 89Zr possesses a relatively
low energy positron (βavg = 395.5 keV), which affords images
agents. Unsurprisingly, the biomedical importance of chelators
with high resolution, the principal driver of its success has been
(organic molecules capable of binding metals) has increased in
its 78.41 h half-life. A half-life of just over 3 days allows images
parallel to this advent of metals in medicine. The clinical use of
to be collected multiple days after injection, making it
chelators is most often associated with treatment for heavy
particularly well-suited for the radiolabeling of IgG antibodies.
metal poisoning, for example, the administration of
Radioimmunoconjugates have a circulation time of several days,
ethylenediaminetetraacetic acid (EDTA) to treat lead poison-
ing.1,2 However, chelators are also critical components of metal-
and longer imaging windows allow for both the accumulation of
the tracer in the target tissue and the clearance of any unbound
containing diagnostics and therapeutics, in particular nuclear
tracer from the blood pool. This in turn leads to improved
imaging agents bearing metallic radioisotopes. In these cases,
the role of the chelator is to stably sequester the radiometal and
prevent its release from the agent. While a number of excellent
chelators for common radiometals are known, the recent
image contrast and tumor-to-background activity ratios.
Yet a radionuclide with suitable decay characteristics is not
sufficient to make an effective imaging agent. It is also necessary
to have a reliable method of chelating the radiometal and
emergence of a few nontraditional medically useful isotopes
attaching the chelate complex to the targeting vector. In the
have increased the demand for novel chelators specifically
tailored to the chemistry of these new radiometals.3,4 The case
of the positron-emitting radiometal zirconium-89 (89Zr)
Received: March 12, 2014
provides a prime example of this phenomenon.
Published: May 9, 2014
© 2014 American Chemical Society
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case of 89Zr, the most commonly used chelator is desferriox-
amine B (DFO, Figure 1). DFO, a natural bacterial side-
has focused on investigating the coordination chemistry of Zr4+
with different hydroxamate binding groups with the goal of
gaining insights toward the design of new chelators.35 Most
notably, this work further confirmed that Zr4+ preferentially
forms eight-coordinate complexes and thus strengthens the
argument for the development of an octadentate chelator.
These data have led us to conclude that a ligand designed
specifically with the chemistry of Zr4+ in mind may demonstrate
improved stability upon complexation with Zr4+. Furthermore
and perhaps even more importantly, a more stable complex
should be less prone to demetalation in vivo, resulting in
reduced bone uptake and thus a safer and more efficient 89Zr-
based PET tracer.
Our approach to designing a chelator for Zr4+ was inspired by
a library of ligands developed by Raymond et al. as actinide
sequestration agents.36 The library contained a number of
ligands designed for plutonium(IV) decorporation,37−39 and
the similarity in coordination chemistry between Zr4+ and Pu4+
marked these as excellent candidates. The use of Hf4+ (the third
row congener of Zr4+) as a model for Pu4+ highlights this
connection. These ligands are composed of hard oxygen donor
groups, specifically hydroxamates, catecholates, and hydrox-
ypyridinonates.36 One ligand in particular, 3,4,3-(LI-1,2-
HOPO) (Figure 1), stood out as a promising candidate for
Zr4+ chelation. This ligand, referred to herein as HOPO, is an
octadentate chelator composed of a spermine backbone
coupled with four hydroxypyridinone groups for metal binding.
The hydroxypyridinone groups offer hard, oxygen donors
appropriate for binding Zr4+ and have pKa values of −0.8 and
5.8 compared to the much higher pKa values of the catecholate,
hydroxamate, and alkylhydroxamate (pKa of 9.5, 9.3, and 8.7,
respectively).35,36,40 Zr4+ has been shown to successfully
compete with Pu4+ in extraction experiments using a resin
bearing 1,2-hydroxypyridinone groups,41 which supports our
hypothesis that the 1,2-hydroxypyridinone moiety would form
a strong chelator for Zr4+. The linear arrangement of donor
atoms should impart faster binding kinetics compared to their
arrangement in a macrocycle. Taken together, the eight-
coordinate binding and the hard oxygen donor groups of the
HOPO ligand should be an ideal coordination environment for
Zr4+ and offer good in vivo stability. While the HOPO ligand is
admittedly not novel, its application to 89Zr4+ and PET is
unique.
Figure 1. Structures of the currently used chelator for 89Zr, DFO, and
the newly investigated alternative chelator, HOPO.
rophore, is a hexadentate ligand with three hydroxamate groups
that provide six oxygen donors (three anionic and three
neutral) for metal binding.26 In addition, DFO has a pendent
amine that has been derivatized in a number of ways to create
bifunctional variants of the chelator for facile conjugation to
antibodies and other biomolecular vectors; for example, one
such derivative is the commercially available benzyl isothiocya-
nate DFO.27 Regardless of which bifunctional DFO derivative is
used for conjugation, DFO-mAb conjugates are typically
radiolabeled with 89Zr under mild conditions (pH 6.5−8.0,
room temp, 1 h).12,28,29 These radiolabeled bioconjugates can
then be purified by either size exclusion chromatography or
centrifugal filtration prior to use.
While DFO is currently the gold standard for 89Zr chelation,
there is certainly room for improvement. The primary issue
with DFO is that even purified 89Zr-DFO-mAb conjugates have
been shown to produce significant uptake of radioactivity in the
bones of mice, typically on the order of 10 %ID/g.14,19,30−32
This uptake is not the result of the radioimmunoconjugate as a
whole. Rather, the free 89Zr4+ cation is known to be osteophilic,
meaning the metal itself is readily mineralized into the
skeleton.28,33 Therefore, it becomes clear that 89Zr4+ is being
released from its chelator within the body. This uptake of 89Zr4+
is of particular concern in the clinic, for accumulation of 89Zr4+
in the bone can dramatically increase radiation dose to the bone
marrow, an especially radiosensitive tissue. This concern over in
vivo stability has led several groups to investigate the possibility
of developing a better chelator for 89Zr.
From an inorganic chemistry perspective, DFO is not ideally
suited to the coordination properties of Zr4+. The Zr4+ cation is
a highly charged, hard Lewis acid with a predilection for
forming complexes with high coordination numbers. For
example, eight-coordinate complexes such as zirconium oxalate,
[Zr(oxalate)4]4−, are particularly common with Zr4+.34 With
this in mind, it seems incongruous that a hexadentate ligand
such as DFO is used with a metal that prefers forming
octacoordinate complexes. In fact, previous molecular modeling
of the most favorable structure of the Zr-DFO complex
revealed that in the optimized structure, two water molecules
join the metal’s coordination sphere to form an eight-
coordinate complex.14 Preliminary work by Brechbiel et al.
In this study, we investigate the synthesis and character-
ization of the HOPO ligand and the Zr-HOPO complex as well
as the radiolabeling, characterization, stability, and in vivo
behavior of 89Zr-HOPO in comparison to 89Zr-DFO. The
comparative stabilities are further investigated by density
functional theory (DFT) calculations. Taken together, these
studies demonstrate the great potential of 3,4,3-(LI-1,2-
HOPO) and that family of ligands as chelators for 89Zr4+.
RESULTS AND DISCUSSION
■
An ideal chelator for Zr4+ must meet a few core requirements:
(1) it must be octadentate to fully saturate the coordination
sphere of Zr4+; (2) it must have hard oxygen donors to
complement the hard, oxophilic Zr4+ cation; (3) it must offer
an appropriate sized cavity for the 0.84 Å effective ionic radius
of Zr4+.42 Additionally, an acyclic chelator would be preferred in
order to facilitate faster, more efficient radiolabeling conditions.
It was with these factors in mind that the HOPO ligand was
chosen for investigation as an alternative, more effective
chelator for Zr4+.
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Synthesis and Characterization. The HOPO ligand was
synthesized as previously reported31,36,43−45 (Scheme S1).
However, the adaptation of this ligand for radiochemical
applications required additional purification stringency in order
to eliminate the possibility of preferentially labeling trace
impurities. Therefore, for the investigation at hand, the final
product was purified via reverse-phase HPLC.
The high degree of symmetry of HOPO, combined with its
flexibility, resulted in a 1H NMR with many overlapping
multiplets due to different conformers of the ligand. As a result,
the classification of HOPO as one compound with confounding
conformers rather than distinct isomers or impurities was based
on the identification of the expected compound by mass
spectrometry as well as the appearance of a single peak by
HPLC analysis (Figures S1 and S2).
nonradioactive, characterized Zr-HOPO species. HPLC also
confirmed the purity of both the HOPO ligand and the Zr-
HOPO complex to be higher than 95%. Further character-
ization of the Zr-HOPO complex was attempted using
ultraviolet−visible (UV−vis) spectroscopy; however, the results
were inconclusive as the spectral changes associated with the
ligand−metal complexation were minimal (Figures S8 and S9).
DFT Calculations. The difficulties intrinsic to studying the
aqueous chemistry of Zr, and these Zr-ligand complexes in
particular, preclude the use of potentiometric titrations or
extensive NMR studies because of solubility issues. Therefore,
in order to further investigate the coordination chemistry of the
Zr-ligand complexes, we performed density functional theory
(DFT) calculations on both Zr-HOPO and Zr-DFO. Addi-
tionally, the results of the Zr-HOPO calculations led us to
investigate a slightly modified HOPO ligand adapted to
overcome an observed weakness in the Zr-HOPO structure.
DFT calculations were performed using the Gaussian 0948 suite
of computer programs. All calculations used the CEP-121G
basis set and the B3LYP functional. We tested four basis sets
contained in Gaussian 09 that can treat Zr (CEP-121G, CEP-
31G, LanL2DZ, and QZVP), for their accuracy treating the gas
phase acidity of two simple alcohols (ethanol and methanol) as
a test of their abilities to accurately treat the organic ligands
(which are polyalcohols). CEP-121G and QZVP performed the
best (and about equally well). We chose CEP-121G, as it used
less computational resources. The detailed data can be found in
Table S1. The geometries of all the species were completely
optimized in all internal degrees of freedom while considering
several different conformations of the ligands and complexes.
We base our reports on the lowest energy conformations. The
vibrational frequencies on the CP-corrected potential energy
surface confirmed all structures to be minima (as all frequencies
are real) and enabled the calculation of enthalpies at 298 K,
using the normal harmonic approximations employed in the
Gaussian 09 program.48 The counterpoise correction for basis
set superposition error49−52 was incorporated via optimization
on the CP-corrected potential energy surfaces (CP-OPT)52 in
which two fragments were considered (the Zr and all ligands as
a single fragment) for the gas phase calculations. We used these
frequencies to calculate the IR spectrum of Zr-HOPO (Figure
S7) using a scaling factor of 0.992 which we obtained for the
B3LYP/CEP-121G by comparing the CO stretch of N-
methylacetamide with the experimental value,53 as we have
done previously for B3LYP/D95**.54 We used the Gauss-
View55 program to visualize the spectrum, choosing a
resolution of 15 cm−1 for the best match to the experimental
spectrum.
The nonradioactive Zr-HOPO complex was formed on the
macroscopic scale to facilitate characterization. The complex
was formed by mixing the HOPO ligand with a slight excess
(1.5 equiv) of ZrCl4 in water at room temperature. The Zr-
HOPO product was analyzed by HPLC and eluted as a single
peak with a retention time shifted about 30 s compared to the
ligand alone (Figure S3). The 1:1 binding of Zr4+ and HOPO
was confirmed by high resolution mass spectrometry (HRMS),
which shows the expected mass signals (859.12, [M + Na]+)
1
(Figure S4). While the H NMR of the Zr-HOPO complex
contained the same type of complicated overlapping multiplets
as the ligand itself, there were significant changes in the signals
most closely associated with binding of Zr4+ (Figure S5). The
participation of the hydroxypyridinone rings in the complex-
ation of zirconium was confirmed through infrared (IR)
spectroscopy which shows a red shift in the signals
corresponding to the hydroxypyridinone’s carbonyl groups
(from 1630 to 1610, from 1564 to 1537 cm−1, and from 1375
to 1360 cm−1) as a result of the oxygens binding Zr4+ (Figure
S6).46,47 The calculated IR spectra resulting from the DFT
work assign these signals to highly coupled asymmetric
carbonyl stretches (Figure S7).
Next, the radioactive 89Zr-HOPO complex was synthesized
by radiolabeling the HOPO ligand with a neutralized 89Zr
solution at a pH of ∼7.0. The identity of the radioactive 89Zr-
HOPO species was confirmed through a coelution study
wherein both the radioactive and nonradioactive Zr-HOPO
complexes were injected into the HPLC system together
(Figure 2). Their coelution demonstrates that the 89Zr-HOPO
species possesses the same structure and chemistry as the
We used the CPCM polarizable conductor-like solvent
continuum model49,51with the Bondi50 (rather than the default
UFF56) option for the atomic radii used for the cavity as the
model for the aqueous bulk. We have recently found that the
radii used to define the cavities in CPCM have a significant
effect upon the ΔGsolv values of both N-methylacetamide
(NMA) and water.57 The Bondi radii50 generally gave the best
results when the electrostatic part was used alone, as in this
work. We note that CPCM gives ΔGsolv values rather than ΔH
values. For simplicity, we have labeled as ΔH values the
quantities obtained from a normal vibrational correction for
enthalpy calculated using the CPCM Hamiltonian. We used
single point a posteriori CP corrections for these calculations,
as Gaussian 09 does not allow CP-OPT to be used with
solvation models.
Figure 2. HPLC chromatogram of the co-injection of radioactive 89Zr-
HOPO and nonradioactive Zr-HOPO. The ∼30 s separation between
the two peaks is due to the sequential setup of the UV and radioactive
detectors.
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We calculated the geometry optimized structures of four
complexes: (I) the Zr-HOPO complex [Zr4+ + 3,4,3-(LI-1,2-
HOPO)4−]; (II) a complex of an alternative HOPO-based
ligand, where the central aliphatic chain on the backbone
possesses three carbons instead of four [Zr4+ + 3,3,3-(LI-1,2-
HOPO)4−]; (III) a previously studied Zr-DFO complex [Zr4+
+ DFO2− + 2H2O]2+, as well as another conformation of the
same complex that we found to be more stable (IIIa); and (IV)
the DFO and water molecules. Additionally, no correction was
made for basis set superposition error (BSSE). We thought it
prudent to repeat this calculation using the CEP-121G basis set
to provide direct comparison to our calculations. We also
further explored the potential energy surface (PES) of III,
finding a more stable structure, IIIa, that contains H-bonds
from each of the H atoms on the protonated terminal amine to
the oxygens of the DFO (Figure 3). One of the H-bonds is
bifurcated (the H interacts with two different oxygens), a
common behavior for ammonium salts.58 We found the ΔH of
IIIa to be 10.6 kcal/mol lower (gas phase) and 0.9 kcal/mol
higher (aqueous solution using CPCM) than III. The lower
ΔH of III in solution is due to the extended terminal
ammonium that can be more easily solvated than the internally
H-bonded structure of IIIa. Table 1 presents the calculated Zr−
a neutral, uncharged Zr-DFO complex [Zr4+ + DFO2−
+
2OH−] (Figure 3). We considered III and IIIa for comparison
with a previous study on Zr-DFO2+ but also included IV for a
better comparison with I and II, which are both neutral
complexes. All structures were evaluated in the gas phase as
well as with the CPCM to mimic an aqueous environment.
The previously reported DFT study14 of III used two
different basis sets: LANL2DZ for the Zr and 6-31+G(d) for
a
Table 1. DFT Calculated Zr−O Bond Lengths
terminal ligands
central ligands
I
CO−Zr
NO−Zr
CO−Zr
NO−Zr
CO−Zr
NO−Zr
CO−Zr
NO−Zr
CO−Zr
NO−Zr
CO−Zr
NO−Zr
CO−Zr
NO−Zr
CO−Zr
2.215
2.269
2.220
2.266
2.261
2.121
2.277
2.142
2.262
2.197
2.347
2.367
2.222
2.252
2.172
2.158
2.326
2.167
2.298
2.254
2.220
2.284
2.249
2.281
2.142
2.362
2.229
2.222
2.252
2.196
2.205
2.270
2.205
2.263
2.199
2.111
2.211
2.144
2.237
2.188
2.442
2.417
2.212
2.242
2.163
2.283
2.223
2.291
2.218
n/a
II
III (ref 13)
n/a
III
n/a
n/a
IIIa
n/a
n/a
IV
n/a
n/a
Zr(1,2-HOPO)4
2.212
2.242
2.199
Zr(hydroxamate)4
crystal (ref 35)
NO−Zr
2.215
2.233
2.189
2.178
a
All bond lengths reported in Å. DFT calculations were performed
using the Gaussian 09 program with CEP-121G as the basis set.
O bond distances in the five structures detailed above as well as
for Zr chelated by four free 1,2-hydoxypyridinone (1,2-HOPO)
groups (Figure S10). The bond lengths of an X-ray
crystallographic study on a related compound, Zr-
(hydroxamate)4, are also included for reference.9 The Zr−O
distances for the DFO oxygens are all longer than those
previously reported, but those between Zr and the water
oxygens are somewhat shorter (∼0.01−0.03 Å, Table 1).
However, the orientations of the waters in the two structures
are slightly different. The difference might be due to the BSSE
correction and/or the loss of basis set balance, especially across
the Zr−O bonds. One generally expects bond elongation when
optimization is performed on a BSSE-corrected surface, as
noted for the Zr−O bonds in Zr-DFO. We have also noted that
optimization of certain systems (e.g., water dimer59) converge
to incorrect structures when small basis sets are used on a
surface not corrected for BSSE. Because of the steric problems
within the ligand that modification of the Zr-DFO bonds would
provoke, changing the orientations of these oxygens would be
more difficult than those of the waters.
Figure 3. General chemical structures and optimized DFT structures
for (I) the Zr-HOPO complex; (II) a complex of an alternative
HOPO-based ligand [3,3,3-(LI-1,2-HOPO)] with Zr; (III) a
previously studied positively charged Zr-DFO complex; (IIIa) a
new, more stable conformation of the same charged Zr-DFO complex;
and (IV) a neutral, uncharged Zr-DFO complex.
While Zr-HOPO (I) has two more Zr−O interactions than
Zr-DFO (IV), we could not assume it to be more stable, as IV
has two Zr−OH interactions to compensate for the missing
interactions with the ligand. We used reaction 1 to determine
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the relative stability of the two complexes. We note that the
structure of DFOH2 has a protonated amine that hydrogen-
bonds to three oxygens, one of which is not protonated, which
results in a neutral structure (Figure S11).
I + DFOH2 + 2H2O → IV + HOPOH4
(1)
The calculated enthalpies of this reaction are 47.9 kcal/mol
(gas) and 31.8 kcal/mol (CPCM). These data strongly suggest
that Zr-HOPO is more stable than Zr-DFO. The difference
between the gas phase and CPCM enthalpies reflects the large
solvation free energy of the small OH− anion. However, the
difference seems quite large for two species that each have eight
Zr−O interactions. One reason for this large difference appears
to be that Zr−O distances found in IV are about 0.01 Å larger
on average, suggesting these bonds in IV to be weaker than in I
or II. These calculations suggest a remarkable improvement in
energetic favorability for Zr-HOPO over Zr-DFO.
When we optimized the geometry of I, we found several local
minima. Structure I has the lowest energy, but we thought that
we might find a more stable conformation if we eliminated the
unfavorable gauche orientation of the -(CH2)4- linker between
the middle two chelating groups of I. As we could not find a
more stable structure for I, we decided to eliminate one of the
CH2 groups from I to form II: Zr complexed with a new
HOPO-based ligand, 3,3,3-(LI-1,2-HOPO), with a shortened
center spacer in the backbone (Figure 3). We expect that the
modified ligand of II would bind more tightly to the Zr, since it
eliminates the unfavorable gauche interaction. To test this
hypothesis, we calculated the enthalpy of the reaction that
exchanges the ligands of I and II (reaction 2), which we found
to be −2.8 kcal/mol (gas) and −3.2 kcal/mol (CPCM),
suggesting that the equilibrium constant for reaction 2 is ∼200
at 298 K.
Figure 4. Radio-TLC profiles of the 89Zr-HOPO radiolabeling
reaction over time at different concentrations of ligand. Two peaks
are observed, with the first peak (the kinetic product) converting to
the second peak (the thermodynamic product) over time. The initial
area ratio and separation of the two peaks are dependent upon the
concentration of the ligand.
(and at every point from 10 min onward) no free 89Zr4+ is
observed. This suggests that all of the 89Zr4+ is initially
complexed by HOPO in some fashion but then over time
converts to a different isomer or complex without the 89Zr4+
ever being released. Most likely, this means the complex
undergoes an intramolecular rearrangement of some kind.
Upon further investigation, this phenomenon was found to be
concentration-dependent. As the concentration of HOPO
decreased, two things changed: the secondary peak became
increasingly prevalent at early time points, and the two peaks
appeared closer together and became less distinguishable by
radio-TLC.
The observation that the first peak is less prominent at lower
ligand concentrations suggests a possible explanation for the
identity of the first peak: a dimer of two ligands simultaneously
binding one 89Zr atom. As the concentration of the ligand was
decreased, the likelihood of forming such a dimer would also
decrease, and thus, the first peak would be less abundant. With
this explanation, the second peak is most likely the 1:1 89Zr-
HOPO complex that was expected. This reasoning also explains
how the conversion of the first species to the second could take
place without releasing the metal. Converting from a dimer to a
1:1 complex seems like a reasonable transformation, as the
unused binding groups of one ligand simply replace the binding
sites of the second ligand. This hypothesis is supported by the
fact that the HPLC coelution study (see above) was carried out
24 h after the initial radiolabeling so that the sample only
consisted of the second radio-TLC peak. HPLC analysis
showed the compound responsible for the second radio-TLC
peak to elute with the characterized 1:1 nonradioactive Zr-
HOPO complex, thus confirming its identity.
I + 3,3,3‐(LI‐1,2‐HOPO) → II + 3,4,3‐(LI‐1,2‐HOPO)
(2)
Thus, we expect that II would provide even further advantages
over I as a ligand for Zr4+. Accordingly, we are now exploring
this possibility by synthesizing the 3,3,3-(LI-1,2-HOPO) ligand.
Radiolabeling Experiments. Preliminary radiolabeling
experiments demonstrated quantitative radiolabeling of 10
mM HOPO with 89Zr within 10 min at room temperature at
pH 7 in water. Further experimentation revealed that the time
necessary for complete labeling was dependent on ligand
concentration, with lower concentrations requiring more time.
For example, a HOPO concentration of 10 μM required 45 min
to achieve 100% labeling. In general, radiolabeling reactions
were left to incubate for 1 h to ensure quantitative labeling, and
all radiolabeling reactions were monitored by radioactive thin
layer chromatography (radio-TLC).
Radio-TLC analysis of the labeling solutions revealed an
interesting concentration-dependent speciation phenomenon
(Figure 4). At high concentrations of HOPO (e.g., 10 mM),
radio-TLC analysis 10 min after labeling revealed one sharp
peak at the origin with a small bump immediately following it.
Initially, this seemed like an artifact; however, when radio-TLC
analysis is repeated at later time points, this small bump grows
into a second, distinct peak. Over time, the first peak slowly
converts to the second, and 24 h later, only the second peak
remains. These two peaks must represent two different
complexes of 89Zr and HOPO, one that is the initial kinetic
product and one that is the ultimate thermodynamically stable
product. It is significant to note that throughout this conversion
An explanation for the second observation (that the two
peaks get closer together as the ligand concentration decreases)
is somewhat more elusive; however, it is a consistently
observable trend. The major difficulty in elucidating the exact
speciation is that these phenomena seem only to occur at the
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a
Table 2. EDTA Ligand Challenge
a
Radiolabeled ligand complexes were incubated with 100-fold excess EDTA at set pH values at 37 °C for 7 days. The experiment was done in
triplicate. Green shading indicates >99% intact. Yellow indicates 99−90% intact. Orange indicates 90−80% intact, and red indicates <80% intact.
a
Table 3. Metal Competition
% intact starting species by incubation time
1 h
1 d
5 d
7 d
competing metal cation
89Zr-HOPO
89Zr-DFO
89Zr-HOPO
89Zr-DFO
89Zr-HOPO
89Zr-DFO
89Zr-HOPO
89Zr-DFO
Co2+
Cu2+
Fe3+
Ga3+
Gd3+
K+
Mg2+
Ni2+
Zn2+
98.5 2.2
98.2 2.5
97.0 4.2
98.4 2.3
98.2 2.6
99.0 1.5
98.3 2.5
98.4 2.3
98.6 2.0
92.5 0.4
95.8 4.0
90.6 4.5
94.2 5.6
94.3 1.4
97.2 4.0
94.4 0.02
97.2 4.0
92.9 2.2
98.2 2.6
98.1 2.7
89.8 5.4
97.9 3.0
97.8 3.1
98.7 1.9
98.3 2.5
98.4 2.2
98.7 1.9
93.4 3.1
98.2 2.6
48.2 27.0
94.9 4.3
95.0 2.7
94.8 3.5
95.6 2.5
94.9 3.2
97.9 3.0
98.6 2.0
98.3 2.4
90.5 3.8
97.3 1.9
96.5 2.9
98.5 2.2
98.6 2.0
98.7 1.9
97.7 3.3
96.2 0.5
96.8 1.5
51.9 11.2
93.0 4.4
98.4 2.2
97.6 0.7
98.1 0.8
97.0 1.2
97.6 3.4
98.9 1.6
98.4 2.3
83.0 4.2
96.4 0.6
94.5 4.1
98.1 0.7
98.7 1.8
97.4 0.1
98.3 2.4
95.8 1.1
95.3 0.2
39.1 9.9
91.1 2.2
97.0 0.7
97.0 1.6
96.5 0.1
97.8 1.1
96.4 1.3
a
Radiolabeled ligand complexes were incubated with a 10-fold excess of various biologically relevant metal salts in PBS at a pH of 7.4 at 37 °C for 7
days. The experiment was repeated twice.
tracer level. In these experiments, the ligand concentrations are
in the millimolar to micromolar range, while the concentration
of 89Zr is in the nanomolar to picomolar range. These
conditions cannot be mimicked on the macroscale without
inordinate amounts of the HOPO ligand (e.g., ∼3.4 kg of
HOPO for every 10 mg of Zr), so analysis is limited to tests
that can be carried out on the radioactive samples.
Furthermore, as the concentration of the ligand is reduced
and approaches that of the metal, the two peaks move closer
together to the point where they become indistinguishable.
This trend complicates matters because as the two peaks move
ever closer together, they begin to appear to be one peak, yet it
is impossible to know for certain which of the two original
peaks this species represents.
Finally, an alternative explanation for the two peaks is that
they simply represent two different coordination structures of
the 1:1 Zr-HOPO complex. For example, the initial peak may
be HOPO bound to Zr by only six donor groups which
converts to the second peak of full octadentate chelation as the
final HOPO group changes conformation to bind the metal.
While this explanation is consistent with the fact that the
conversion between the two species does not involve releasing
the metal, it does not provide a justification for the
concentration dependence. Further investigations into these
speciation phenomena are ongoing.
Stability Studies. Serum Stability. In order to gain insight
into the in vitro and in vivo kinetic inertness of the Zr-HOPO
complex, serum stability tests were performed to simulate
biological conditions. Both 89Zr-HOPO and 89Zr-DFO
complexes were found to be stable over a 7-day period at 37
°C in human serum (98.8% and 98.3% intact, respectively).
Further, no protein-bound 89Zr was observed in the serum
samples containing the 89Zr-ligand complexes, which confirms
that the complexes remained intact.
EDTA Challenge. Both HOPO and DFO were labeled with
89Zr and then incubated with a 100-fold excess of EDTA at a
range of pH values at 37 °C for a period of 7 days in order to
examine the stability of the Zr-ligand complexes and their
susceptibility to transchelation (Table 2). Analysis of the
samples by radio-TLC allowed for the differentiation of the
complexes, since 89Zr-EDTA migrates along the ITLC strip,
while both 89Zr-DFO and 89Zr-HOPO remain at the origin.
DFO was shown to be surprisingly vulnerable to transchelation,
most notably at lower pH but also at neutral and biological pH.
In contrast, HOPO demonstrated a remarkable resistance to
transchelation, remaining >99% intact over the full 7 days for
the entire range of pH values tested. The fact that a 100-fold
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Figure 5. Coronal PET images of 89Zr-HOPO. Healthy mice were administered 89Zr-HOPO (260 μCi [9.6 MBq] in 0.9% sterile saline) via tail vein
injection and imaged between 10 min and 24 h after injection. The gall bladder (a), gut (b), and bladder (c) can be visualized. The 89Zr-HOPO
complex undergoes rapid renal clearance followed by slower hepatobiliary clearance. No uptake of 89Zr in the bone is observed.
excess of EDTA was unable to strip the 89Zr from HOPO is a
testament to the stability of the complex. In contrast, the
release of 89Zr from DFO suggests that the ligand is susceptible
to transchelation and may experience similar release in vivo to
any number of competing substances in the body. Additionally,
as tumors are known to be more acidic than healthy tissues, the
greatly improved stability of the Zr-HOPO complex over Zr-
DFO at lower pH may prove beneficial for cancer imaging.
Metal Cation Competition. As a counterbalance to the
EDTA challenge that evaluated the potential for another ligand
to outcompete HOPO for the metal cation, a metal
competition study was performed to see if other metal cations
could outcompete 89Zr for the HOPO binding pocket. Both
HOPO and DFO were labeled with 89Zr and then incubated
with a 10-fold excess of various metal salts at pH 7.4 at 37 °C
(Table 3). The samples were monitored for 7 days by radio-
TLC to determine the percentage of intact 89Zr-ligand complex.
Both 89Zr-DFO and 89Zr-HOPO proved to be relatively stable
(>95% intact) when challenged with the majority of competing
metals with the notable exception of Fe3+. In this case, after 7
days of incubation with FeCl3, HOPO released ∼17% of the
89Zr4+ compared with DFO which released >50% of the 89Zr4+.
Since DFO is originally a siderophore, its affinity for iron over
zirconium is unsurprising. DFO also exhibited a slight release of
89Zr4+ in the presence of Ga3+ (∼9%). This competition further
demonstrates the stability of the Zr-HOPO complex and shows
an advantage over Zr-DFO.
excretion of the complex through the gut of the mice but is not
fully understood at present. This explanation is supported by
the fact that the activity in the gall bladder decreases over time.
Importantly, no significant bone uptake was observed in the
imaging at any time point, indicating that there was no release
of the 89Zr4+ cation from the ligand in vivo. For comparison,
previous studies with the 89Zr-DFO complex show only rapid
renal clearance of the radiotracer.14,33,60
Biodistribution. An ex vivo biodistribution study was also
performed in healthy mice to further probe the biological
behavior of the 89Zr-HOPO complex The results of this study
match the trends observed in the PET images, including the
involvement of both the renal and fecal clearance pathways.
(Table 4). At 10 min after injection, the activity was found
largely in the kidneys (9.46 2.71 %ID/g), suggesting rapid
renal excretion, as well as the small intestine (5.99 1.18 %ID/
g) and the gall bladder (6.61 2.87 %ID/g), consistent with
hepatobiliary excretion. By the 1 h time point, most of the
activity had already cleared through the kidneys (1.05 0.51 %
ID/g) but was still seen in the gall bladder (6.94 3.38 %ID/
g). The large intestine contains the majority of the radioactivity
at 4 h (7.17 2.15 %ID/g), while the 12 and 24 h time points
demonstrate the further clearance of the remaining activity
from all of the organs examined. Notably, the low amount of
activity in the bone, which decreases over time (from 1.04
0.44 to 0.17 0.03 %ID/g), is consistent with the clearance of
the intact complex rather than accumulation due to
mineralization of the free radiometal.
In Vivo Studies. An advantage of working with a ligand that
has already been evaluated for a different biological application
is that studies into the toxicity of the compound have already
been carried out.31 The toxicity of HOPO was studied in both
mice43 and baboons,45 and neither showed any evidence of
harm with frequent, repeated injections of the HOPO ligand at
concentrations of 10 and 30 μmol/kg, respectively. These data,
combined with the fact that the extremely small amounts of
compound needed for radiopharmaceutical studies are far
below the threshold for pharmacological effects, allowed for the
immediate evaluation of 89Zr-HOPO in mice without concerns
about adverse reactions.
Imaging. PET imaging studies were performed in order to
investigate the in vivo pharmacokinetics of the 89Zr-HOPO
complex. To this end, the 89Zr-HOPO complex (260 μCi, 9.6
MBq, in 200 μL of 0.9% sterile saline) was injected into healthy
mice and imaged at four time points using PET (Figure 5). At
10 min, the vast majority of the radioactivity is observed in the
bladder, which indicates rapid renal clearance. At the remaining
time points (4, 12, and 24 h), much of the radioactivity is seen
in the gut, signifying hepatobiliary as well as renal excretion.
Interestingly, there was significant activity localized in the gall
bladder: this is most likely a consequence of the hepatobiliary
In order to make direct comparisons, the in vivo
biodistribution of 89Zr-DFO was investigated in the same
study (Figure 6). As expected, 89Zr-DFO cleared exclusively
through the kidneys and thus was eliminated more quickly than
89Zr-HOPO. The fact that 89Zr-DFO does not show any gall
bladder uptake further suggests that the uptake of 89Zr-HOPO
in the gall bladder is a result of hepatobiliary excretion. Other
than the difference in excretory pathways, the clearance of the
two complexes through the blood and other organs appears
similar, with neither complex accumulating appreciably any-
where in the body.
There is a statistically significant difference in the amount of
activity in the bone between 89Zr-DFO and 89Zr-HOPO, with
higher levels observed for 89Zr-HOPO. This could be the result
of the release of the 89Zr4+ cation from the HOPO ligand due
to instability or metabolism; however, if this were the case, we
would expect to see a more substantial amount of radioactivity
in the bone and for the amount of activity to stay constant or
grow over time. Instead, with such a small amount of activity
and no evidence of long-term accumulation of the radiometal in
the bone, this is most likely a question of the perfusion and
clearance kinetics of 89Zr-HOPO rather than demetalation.
Since 89Zr-DFO is only cleared renally and not through the
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Figure 6. Biodistribution of 89Zr-HOPO and 89Zr-DFO in select
organs. Healthy, athymic nude mice were injected with either 89Zr-
HOPO or 89Zr-DFO (24−35 μCi [0.89−1.29 MBq] in 0.9% sterile
saline) via the tail vein, sacrificed at specified time points, and
necropsied. The concentration of radioactivity in the chosen organs is
expressed as %ID/g and presented as an average value from four
animals standard deviation. Bl = blood, GB = gall bladder, L = liver,
LI = large intestines, SI = small intestines, K = kidney, Bo = bone. ∗
indicates P values of <0.05.
hepatobiliary system, it is excreted from the body faster than
89Zr-HOPO, as evidenced by the blood clearance curve (Figure
7). We postulate that just as 89Zr-HOPO takes a longer time to
clear the blood, it also takes a longer time to clear the bone.
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the 89Zr-HOPO complex to have good biological behavior,
clearing the body cleanly without any signs of demetalation.
The ultimate test of the efficacy of the HOPO ligand will
come when the ligand can be conjugated to an antibody,
radiolabeled, and tested in vivo in the context of its desired
application. To this end, bifunctionalization of the HOPO
ligand is currently underway, and evaluation of the ligand when
conjugated to an antibody will be forthcoming once the
synthesis is complete. Additionally, the 3,3,3-(LI-1,2-HOPO)
ligand is also being synthesized to follow-up on the intriguing
results of the DFT calculations which suggest that a shortened
backbone will impart even greater stability upon binding of
Zr4+. For now, the HOPO ligand shows tremendous promise as
a chelator for 89Zr and may eventually supplant DFO for
immunoPET applications.
Figure 7. Blood clearance of 89Zr-HOPO and 89Zr-DFO in healthy,
athymic nude mice (n = 4) over time. Inset shows a zoomed graph for
further detail.
EXPERIMENTAL SECTION
■
Materials and Methods. All chemicals, unless otherwise noted,
were acquired from Sigma-Aldrich (St. Louis, MO) and used as
received without further purification. Human serum was purchased
frozen from Sigma-Aldrich. All water used was ultrapure (>18.2 MΩ
cm−1). All instruments were calibrated and maintained in accordance
with standard quality-control procedures. High resolution mass
spectrometry (HRMS) measurements were performed on a Waters
SYNAPT high definition MS system (ESI-QTOF) and low resolution
mass spectra were recorded with a Waters Acquity UPLC with
electrospray ionization (ESI) SQ detector. 1H NMR spectra were
recorded at ambient temperature on a Bruker 500 MHz NMR with
Topsin 2.1 software. The NMR spectra are expressed on the δ scale
and were referenced to residual solvent peaks and/or internal
tetramethylsilane. The HPLC system used for analysis and purification
compounds consisted of a Shimadzu prominence system equipped
with a diode array and a radioactive detector (Bioscan Inc.,
Washington, DC). IR spectroscopy was performed on a solid sample
using an attenuated total reflectance attachment on a PerkinElmer
Spectrum 2 FT-IR spectrometer with a UATR Two attachment. UV−
vis spectroscopy was carried out using a Thermo Scientific Evolution
220 UV−visible spectrophotometer.
The higher levels of 89Zr measured in the bone are likely a
result of higher levels of 89Zr still present in the mouse.
The difference in clearance pathways between 89Zr-HOPO
and 89Zr-DFO should not be a concern in the long run. Upon
conjugation to an antibody, the pharmacokinetics of the 89Zr-
HOPO complex will be completely superseded by those of the
biomacromolecule. However, at this point in the development
of the ligand, with only the bare 89Zr-ligand complex available,
it is only possible to test its in vivo stability for the
comparatively short circulation time associated with the small
molecule complex as opposed to the several day circulation
time of a radioimmunoconjugate. Therefore, while the in vivo
stability of the 89Zr-HOPO complex demonstrated here is very
promising, it alone is insufficient to prove its viability in 89Zr-
HOPO-based radioimmunoconjugates. With this in mind, the
longer duration in vitro studies were intended to supplement
the in vivo experiments by allowing us to probe the longer
stability requirements of the antibody labeling applications. In
sum, the combination of the in vitro and in vivo experiments
gives a more complete picture of the stability of the 89Zr-
HOPO complex and illustrates the great potential of the
HOPO ligand.
89Zr was produced at Memorial Sloan-Kettering Cancer Center on a
TR19/9 cyclotron (Ebco Industries Inc.) via the 89Y(p,n)89Zr reaction
and purified to yield 89Zr with a specific activity of 196−496 MBq/mg.
Activity measurements were made using a CRC-15R dose calibrator
(Capintec). For the quantification of activities, experimental samples
were counted on an Automatic Wizard (2) g-Counter (PerkinElmer).
The radiolabeling of ligands was monitored using salicylic acid
impregnated instant thin-layer chromatography paper (ITLC-SA)
(Agilent Technologies) and analyzed on a Bioscan AR-2000 radio-
TLC plate reader using Winscan Radio-TLC software (Bioscan Inc.,
Washington, DC). All in vivo experiments were performed according
to protocols approved by the Memorial Sloan-Kettering Institutional
Animal Care and Use Committee (Protocol 08-07-013). Purity of
greater than 95% was confirmed using quantitative HPLC analysis for
nonradioactive compounds (HOPO and Zr-HOPO) and radio-TLC
for radioactive compounds (89Zr-HOPO).
CONCLUSIONS
■
In terms of Zr4+ chelation and stability, 3,4,3-(LI-1,2-HOPO)
has proven to be a superior ligand compared to DFO. We have
synthesized the HOPO ligand, synthesized and characterized
the Zr-HOPO complex, and evaluated the stability and
behavior of the 89Zr-HOPO complex both in vitro and in
vivo. HOPO has proven comparable and often superior to
DFO in every test we have conducted, both experimentally and
computationally. Our DFT calculations have shown Zr4+
complexed with HOPO to be significantly more stable than
with DFO and have suggested that the complex with 3,3,3-(LI-
1,2-HOPO), a modified hydroxypyridinone-based ligand, might
be even more stable still. The improved stability of the 89Zr-
HOPO complex is most dramatically shown in the EDTA
challenge experiments in which the 89Zr-HOPO complex is able
to resist transchelation far better than 89Zr-DFO. This increase
in stability is especially significant because it is observed not
only at lower pHs like 5.0−6.5 at which the lower pKa of the
hydroxypyridinone was an expected advantage but also at
biologically relevant pHs like 7.0−7.5 at which DFO was still
susceptible to partial transchelation. In addition, we have shown
3,4,3-(LI-1,2-HOPO) (HOPO). The ligand was synthesized as
previously described with slight modification. The details are presented
in the Supporting Information.
Zr-HOPO. A solution of zirconium chloride (1.5 mg, 6.0 μmol) in
water (0.5 mL) was added to a solution of HOPO (3.0 mg, 4.0 μmol)
in water (0.5 mL). The mixture was vortexed and left for 15 min. The
resulting solution was cloudy with some white precipitate. The
complex was not very soluble in water; however, enough compound
remained in solution to allow for analysis of the complex via HRMS
and HPLC. NMR analysis remained difficult because of low solubility
as well as the indistinct, overlapping multiplets of the HOPO ligand
itself (Figure S5). A solution of nonradioactive Zr-HOPO was
lyophilized, and the resulting off-white powder was analyzed by IR
spectroscopy. Characterization of the Zr-HOPO complex by UV−vis
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spectroscopy was attempted but not conclusive. The details are
presented in the Supporting Information.
89Zr-ligand complexes in competing metal solutions were put in a heat
block at 37 °C to incubate with agitation. Sample mixtures were
checked by radio-TLC after 1 h, 1 d, 5 d, and 7 d. Experimental data
represent the averages and standard deviations of two sets of
experiments.
Radiolabeling Studies. 89Zr was received after target processing
as 89Zr oxalate in 1.0 M oxalic acid. This solution is then neutralized
with 1.0 M sodium carbonate to reach pH 6.8−7.2. Both the DFO and
HOPO ligands were labeled at various concentrations in water or
saline with the neutralized 89Zr solution at room temperature for
varying lengths of time, typically 10−60 min. Samples were labeled
with 10 μCi to 1 mCi depending on the nature of the experiment (for
reference, 1 μCi 89Zr equals 25 fmol). Reactions were monitored via
radio-TLC using Varian ITLC-SA strips (Agilent Technologies) and
50 mM EDTA at pH 5 as the mobile phase. 89Zr-ligand complexes
remained at the origin, while free 89Zr was taken up by EDTA in the
mobile phase and migrated along the ITLC strip.
PET Imaging. PET imaging experiments were conducted on a
microPET Focus 120. Healthy female, athymic nude mice were
administered 89Zr-HOPO (9.6 MBq [260 μCi] in 200 μL of 0.9%
sterile saline) via intravenous tail vein injection (t = 0). Approximately
5 min prior to the acquisition of PET images, mice were anesthetized
by inhalation of 2% isoflurane (Baxter Healthcare, Deerfield, IL)/
oxygen gas mixture and placed on the scanner bed; anesthesia was
maintained using 1% isoflurane/gas mixture. PET data for each mouse
were recorded via static scans at various time points between 10 min
and 24 h. An energy window of 350−700 keV and a coincidence
timing window of 6 ns were used. Data were sorted into 2D
histograms by Fourier rebinning, and transverse images were
reconstructed by filtered back-projection (FBP) into a 128 × 128 ×
63 (0.72 × 0.72 × 1.3 mm3) matrix. The image data were normalized
to correct for nonuniformity of response of the PET, dead-time count
losses, positron branching ratio, and physical decay to the time of
injection, but no attenuation, scatter, or partial-volume averaging
correction was applied. The counting rates in the reconstructed images
were converted to activity concentrations (percentage injected dose
per gram of tissue, %ID/g) by use of a system calibration factor
derived from the imaging of a mouse-sized water-equivalent phantom
containing 89Zr. Images were analyzed using ASIPro VM software
(Concorde Microsystems).
Biodistribution. Acute in vivo biodistribution studies were
performed in order to evaluate the uptake of the 89Zr-HOPO and
89Zr-DFO in healthy female, athymic nude mice. Mice were warmed
gently with a heat lamp for 5 min before administration of 89Zr-HOPO
(0.89−1.11 MBq [24−30 μCi] in 200 μL of 0.9% sterile saline) or
89Zr-DFO (1.11−1.29 MBq [30−35 μCi] in 200 μL of 0.9% sterile
saline) via intravenous tail vein injection (t = 0). Animals (n = 4 per
group) were euthanized by CO2(g) asphyxiation at 10 min, 1 h, 4 h, 12
h, and 24 h. After asphyxiation, 14 organs were removed, rinsed in
water, dried in air for 5 min, weighed, and counted in a γ counter
calibrated for 89Zr. Counts were converted into activity using a
calibration curve generated from known standards. Count data were
background- and decay-corrected to the time of injection, and the
percent injected dose per gram (%ID/g) for each tissue sample was
calculated by normalization to the total activity injected. The full data
set of organs is included in the Supporting Information (Table S2)
along with values represented in %ID without normalization (Table
S3). Biodistribution data were assessed by unpaired t tests using
GraphPad Prism (version 6.02 for Windows GraphPad Software, San
Diego, CA, U.S.) in order to determine any significant differences (p <
0.05) (Table S4).
Serum Stability Study. 89Zr-HOPO and 89Zr-DFO were prepared
according to the radiolabeling protocol as described above. For each
89Zr complex, as well as for free, unchelated 89Zr, solutions were made
consisting of 1 mL of human serum and 100 μL of the 89Zr species and
were placed in a heat block at 37 °C with agitation. Samples were
monitored using radio-TLC immediately after mixing and then daily
for 1 week. Intact 89Zr-ligand complexes remained at the origin of the
ITLC strip, while free 89Zr was either bound by serum proteins or
picked up by EDTA in the mobile phase and migrated along the ITLC
strip. The stability of the complexes was measured as the percentage of
89Zr that was retained at the origin of the ITLC strip and therefore still
bound to the chelator. Free 89Zr incubated in serum appeared on
radio-TLC as a series of messy, broad peaks ranging from the origin to
the solvent front. This was likely a result of 89Zr being bound to a
variety of serum components with different retention factors. In order
to confirm that the sharp peaks at the origin seen in the 89Zr-ligand
samples were in fact the 89Zr-ligand complexes and not protein-bound
89Zr, size exclusion chromatography (SEC) was performed on the
serum samples after 7 days (Figure S12). SEC was carried out with a
hand-packed column (18 mm × 180 mm) of Superdex 200 prep grade
gel filtration medium (GE Healthcare) on a BioLogic LP automated
liquid chromatography system (BioRad) with a flow rate of 1 mL/min
for 45 min using PBS as the eluant. Serum protein components were
observed in the UV chromatogram (280 nm), while 89Zr species were
tracked by γ-counting collected 1 mL fractions. Both 89Zr-ligand serum
samples eluted at ∼28 min, which corresponds to the retention time of
the pure 89Zr-ligand controls. The control sample of free 89Zr in
serum, however, eluted at ∼20 min, which corresponds to the
molecular weight of a major serum protein component as seen in the
UV chromatogram.
EDTA Challenge Study. The 100 μL samples were prepared
consisting of 100 μM ligand to be tested (HOPO or DFO). An
amount of 100 μL of 89Zr solution (∼100 μCi) at pH 7 was added to
each sample. Each sample was then spotted onto an ITLC strip,
developed, and analyzed to obtain an initial measure of the percent
labeling. The samples were left to incubate at room temperature for 1
h. Following incubation, 200 μL of 5 mM EDTA at different pH (5.0,
5.5, 6.0, 6.5, 7.0, 7.5, and 8.0) was added to the samples. Lastly, an
amount of 100 μL of 500 mM acetate buffer was added to each sample
to maintain the experimental pH of the solutions. The final
composition of the samples was 500 μL total volume, ∼100 μCi
89Zr, 100 mM acetate buffer, and a 1:100 ratio of ligand/EDTA. The
samples were then transferred to a heat block at 37 °C to incubate for
7 days with agitation. Samples were monitored by radio-TLC at 1 h, 3
h, 1 d, 3 d, 5 d, and 7 d postincubation. Three samples were prepared
at each pH to obtain triplicate data for statistics.
ASSOCIATED CONTENT
* Supporting Information
■
S
Detailed data and figures from ligand and complex character-
ization, DFT calculations, serum stability studies, mouse
imaging, and biodistribution studies. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
*J.J.D.: phone, 212-772-5343; e-mail, jdannenberg@gc.cuny.
■
Metal Cation Competition Study. The 1 mL solutions of 200
μM HOPO and DFO were prepared and radiolabeled with 1 mL (30−
50 μCi) of neutralized 89Zr each. Samples were left to incubate at
room temperature for 1 h before being monitored by radio-TLC to
confirm complete labeling. The 89Zr-ligand samples were then split
into 200 μL aliquots and added to 200 μL solutions of prepared 1 mM
metal salts [cobalt(II) chloride, copper(II) chloride, iron(III) chloride,
gallium(III) nitrate, gadolinium(III) chloride, potassium carbonate,
magnesium chloride, nickel(II) acetate, and zinc acetate]. Samples of
edu.
*J.S.L.: phone, 646-888-3038; e-mail, lewisj2@mskcc.org.
*L.C.F.: phone, 212-772-5353; e-mail, lfrances@hunter.cuny.
edu.
Author Contributions
All authors have given approval to the final version of the
manuscript.
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Article
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Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
The authors thank Blesida Punzalan and Kuntal Sevak for
skillful tail-vein injections, Amandeep Saini for assistance in
carrying out radio-TLC experiments, Dr. Ben Burton-Pye for
spectroscopic advice, and Dr. Xinxu Shi, Dr. David Mootoo,
Ahmad Altiti, and Dr. Malleswari Ponnala for early synthetic
assistance. The authors are grateful to Professor Ken
Czerwinski for helpful discussions. Services provided by the
MSKCC Small-Animal Imaging Core Facility were supported
in part by NIH Grants R24 CA83084 and P30 CA08748. We
gratefully acknowledge the CTSC grant UL1TR000457 of the
National Center for Advancing Translational Sciences of the
National Institutes of Health. The authors also thank the NIH
(Award 1F31CA180360-01, M.A.D.), the NSF (Award IGERT
0965983, L.C.F.), the DOD (Award CDMRP W81XWH-12-1-
0029, B.M.Z.), and the DOE (Award FG02-09ER16097, L.C.F.,
and Award DE-SC0002184, J.S.L.) for their generous funding.
Research infrastructure at Hunter College is partially supported
by Grant Number MD007599 from the National Institute on
Minority Health and Health Disparities (NIMHD) of the NIH.
ABBREVIATIONS USED
■
DFO, desferrioxamine B; HOPO, 3,4,3-(LI-1,2-HOPO); radio-
TLC, radioactive thin layer chromatography; SEC, size
exclusion chromatography
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