H4octapa-trastuzumab: Versatile acyclic chelate system for 111In and 177Lu imaging and therapy

Article abstract of DOI:10.1021/ja4049493

A bifunctional derivative of the versatile acyclic chelator H ₄octapa, p-SCN-Bn-H₄octapa, has been synthesized for the first time. The chelator was conjugated to the HER2/neu-targeting antibody trastuzumab and labeled in high radioch

Full text of DOI:10.1021/ja4049493

Article
pubs.acs.org/JACS
111
H4octapa-Trastuzumab: Versatile Acyclic Chelate System for In
177
and Lu Imaging and Therapy
Eric W. Price,†,§ Brian M. Zeglis,‡ Jacqueline F. Cawthray,†,§ Caterina F. Ramogida,†,§ Nicholas Ramos,‡
Jason S. Lewis,*,‡ Michael J. Adam,*,§ and Chris Orvig*,†
†Medicinal Inorganic Chemistry Group, Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver,
British Columbia, Canada, V6T 1Z1
§TRIUMF, 4004 Wesbrook Mall, Vancouver, British Columbia, Canada, V6T 2A3
‡Department of Radiology and Program in Molecular Pharmacology and Chemistry, Memorial Sloan-Kettering Cancer Center, 1275
York Avenue, New York, New York, 10065, United States
S
* Supporting Information
ABSTRACT: A bifunctional derivative of the versatile acyclic
chelator H4octapa, p-SCN-Bn-H4octapa, has been synthesized
for the first time. The chelator was conjugated to the HER2/neu-
targeting antibody trastuzumab and labeled in high radio-
chemical purity and specific activity with the radioisotopes 111In
and 177Lu. The in vivo behavior of the resulting radio-
immunoconjugates was investigated in mice bearing ovarian
cancer xenografts and compared to analogous radioimmuno-
conjugates employing the ubiquitous chelator 1,4,7,10-tetraaza-
cyclododecane-1,4,7,10-tetraacetic acid (DOTA). The H4octapa-
trastuzumab conjugates displayed faster radiolabeling kinetics
with more reproducible yields under milder conditions (15 min,
RT, ∼94−95%) than those based on DOTA-trastuzumab (60
min, 37 °C, ∼50−88%). Further, antibody integrity was better
preserved in the 111In- and 177Lu-octapa-trastuzumab constructs, with immunoreactive fractions of 0.99 for each compared to
0.93−0.95 for 111In- and 177Lu-DOTA-trastuzumab. These results translated to improved in vivo biodistribution profiles and
SPECT imaging results for 111In- and 177Lu-octapa-trastuzumab compared to 111In- and 177Lu-DOTA-trastuzumab, with
increased tumor uptake and higher tumor-to-tissue activity ratios.
days), and 177Lu, a reactor produced therapeutic radiometal
INTRODUCTION
■
(
176Lu(n, γ)177Lu) that emits β− particles as well as γ-rays (t1/2
Radiometalated bioconjugates possess an inherent modularity
that can prove extremely useful in the design and construction
of agents for nuclear imaging and therapy. Indeed, they contain
a set of four distinct chemical components that can be swapped
systematically to tune the properties and functions of the
whole: the radiometal can be exchanged to harness isotopes
with different decay characteristics; the chelator can be altered
to accommodate different radiometals; the nature of the
covalent linkage between the chelator and the biomolecule can
be changed to take advantage of different types of conjugation
chemistry; and, of course, the biomolecule itself can be replaced
in order to change the targeting properties or pharmacokinetics
of the construct.1−7 In clinical practice, the use of different
isotopes with a single targeting vector becomes especially
important in pretherapy imaging and dosimetry studies,
procedures in which an agent bearing an imaging radioisotope
is used for scouting scans prior to radiotherapy using the same
vector labeled with a therapeutic nuclide. Two isotopes that are
ideally suited for this purpose are 111In, a cyclotron produced
radiometal (111Cd(p,n)111In) for SPECT imaging (t1/2 ∼2.8
∼6.6 days).8
Due to their exceptional specificity and affinity, antibodies
have emerged as extremely promising biomolecules for the
delivery of radioactive payloads to cancerous tissues.9,10
However, antibodies are not without their limitations as
radiopharmaceutical vectors. For example, while they are
generally considered robust in vivo, antibodies can become
damaged or break down when subjected to the elevated
temperatures required by many radiometal chelation reac-
tions.11 As a result, the choice of a chelator for a radio-
immunoconjugate becomes particularly critical. In any radio-
pharmaceutical, the purpose of the chelator is to sequester the
radiometal such that the isotope remains attached to the
targeting vector at all times, resisting spontaneous demetalation
as well as transchelation to serum proteins. Macrocyclic
chelators, such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra-
Received: May 16, 2013
Published: July 31, 2013
© 2013 American Chemical Society
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a
Chart 1. Structures of Some Selected Chelators
a
Chelators used or discussed in this work, including two based on the pyridinecarboxylate scaffold that were developed in our laboratory: the
promising 67/68Ga chelator H2dedpa and H4octapa, the nonbifunctional variant of the chelator p-SCN-Bn-H4octapa used in this work.
acetic acid (DOTA; Chart 1), have long been staples of
radiochemical research. They are versatile and are generally
more kinetically inert than acyclic chelators, primarily due to
their constrained geometries and partially preorganized
coordination sites.12−19 One unfortunate and restrictive
property of macrocycles like DOTA, however, is that heating
(40−90 °C) and extended reaction times (30−180 min) are
typically required for quantitative radiolabeling, conditions that
can be incompatible with antibodies and other biomole-
cules.12−26 Acyclic chelators offer improvements in this regard,
but not without cost. The acyclic chelator diethylenetriamine-
tetraacetic acid (DTPA), for example, exhibits much faster
reaction kinetics than DOTA and can radiolabel with a variety
of radiometals quantitatively in a matter of minutes at ambient
temperature. However, it is not nearly as stable in vivo as its
macrocyclic counterparts.14,15,27−29
Thus, choosing a radiometal chelator for an antibody
conjugate requires a delicate balancing act. An immunoconju-
gate bearing a macrocyclic chelator, such as DOTA, can require
extended incubations at elevated temperaturesoften at the
expense of antibody stability or immunoreactivityin order to
achieve suitable radiochemical yields; however, a radio-
immunoconjugate bearing an acyclic chelator, such as DTPA,
risks the unintentional release of the radiometal in vivo, an
especially pressing problem considering the long pharmacoki-
netic half-life of many antibodies.8,10,19−22,26−28 It thus becomes
clear that a versatile chelator that combines the thermodynamic
stability and kinetic inertness of macrocyclic chelators with the
facile radiolabeling of acyclic chelators could prove tremen-
dously valuable in the synthesis and development of novel
radioimmunoconjugates.
on the pyridinecarboxylate scaffold (Chart 1).30−38 Members of
the versatile “pa family” of chelators, including H4octapa,
H2dedpa, H5decapa, and H2azapa, cover nearly all of the
current medicinally relevant radiometals.30−38 H4octapa specif-
ically has demonstrated high in vitro and in vivo stability and
rapid radiolabeling kinetics with 111In. Further, comparisons
with 111In-DOTA have revealed that H4octapa possesses much
more facile radiolabeling kinetics, can be labeled in higher
specific activity, undergoes less fluxional isomerization in
solution, and exhibits comparable stability in vitro and in
vivo.33 A major hurdle during this work, however, has been the
cumbersome syntheses of the chelators.30−36 As a result, in the
investigation at hand, we report the construction of the
bifunctional p-SCN-Bn-H4octapa via a vastly improved
synthetic protocol. More importantly, we have employed the
HER2/neu-targeted antibody trastuzumab as a model system
for the in vitro and in vivo validation of 111In-octapa- and 177Lu-
octapa-based radioimmunoconjugates in a murine model of
ovarian cancer as well as the comparative evaluation of these
radioimmunoconjugates to those employing the more tradi-
tional macrocyclic chelator DOTA.
RESULTS AND DISCUSSION
■
Synthesis and Characterization. In order to properly
evaluate the potential of H4octapa for use in biomolecular
radiopharmaceuticals, we first sought to develop a highly
efficient synthesis of a novel bifunctional variant of the versatile
acyclic chelator, p-SCN-Bn-H4octapa (Scheme 1), which had
never been synthesized before. Like many bifunctional
chelators, we have utilized the versatile and enantiopure
starting material L-4-nitrophenylalanine.29,30,33 While the N-
benzyl-based protecting group chemistry we have previously
reported for the synthesis of nonbifunctional H4octapa proved
adequate for the construction of the bare chelator, the requisite
vigorous hydrogenation conditions resulted in poor yields and
were completely incompatible with the synthesis of bifunctional
derivatives based on 4-nitrophenylalanine.30,33 Consequently,
Herein, we report the synthesis, characterization, and in vivo
validation of p-SCN-Bn-H4octapa, a bifunctional, versatile,
acyclic chelator that can be labeled rapidly at room temperature
(RT) and exhibits significant thermodynamic stability and
kinetic inertness with 111In and 177Lu. This ligand has evolved
out of our laboratory’s extensive study of acyclic chelators based
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a
Scheme 1. Improved Synthesis of Chelators Utilizing Nosyl Protection Chemistry
a
(a): (i) THF, NaHCO3, 2-nitrobenzenesulfonyl chloride (2.2 equiv), RT, 24 h, 87%; (ii) DMF, Na2CO3, methyl-6-bromomethylpicolinate (2.2
equiv), 50 °C, 24 h, 95%; (iii) THF, thiophenol (2.2 equiv), K2CO3, RT, 72 h, 91%; (iv) MeCN, Na2CO3, tert-butylbromoacetate (2.2 equiv), 50 °C,
24 h, 92%; (v) 6 M HCl, reflux, 24 h, 66%. (b) (i) THF, NaHCO3, 2-nitrobenzenesulfonyl chloride (2.2 equiv), 50 °C, 24 h, 74%; (ii) DMF,
Na2CO3, tert-butylbromoacetate (2.2 equiv), 50 °C, 24 h, 88%; (iii) THF, thiophenol (2.2 equiv), K2CO3, RT, 72 h, 89%; (iv) MeCN, Na2CO3,
methyl-6-bromomethylpicolinate (2.2 equiv), 50 °C, 24 h, 96%; (v) 5 mL of (1:1) glacial acetic acid:3 M HCl, Pd/C (20 wt %), H2 (g) balloon, RT,
1 h; (vi) 6 M HCl, reflux, 24 h; (vii) thiophosgene in DCM (15 equiv), 3 M HCl, RT, 24 h, 50% over steps v−vii.
we revamped the synthesis to utilize the 2-nitrobenzenesulfo-
namide (nosyl) protecting group, a change that has significantly
improved the synthesis of H4octapa (5) and has made the
synthesis of p-SCN-Bn-H4octapa (12) feasible.30,33,36,39−41
Specifically, the switch to the nosyl protecting group has
dramatically increased the cumulative yield of H4octapa (5)
from ∼10−12% to ∼45−50% over five synthetic steps.33
Further, using this strategy, the bifunctional variant p-SCN-Bn-
H4octapapreviously inaccessible using N-benzyl protection
chemistrycan now be routinely produced with cumulative
yields over 7 steps of ∼25−30%. To our knowledge, only a few
previous publications have utilized the 2-nitrobenzenesulfona-
mide (nosyl) amine-protecting group for the synthesis of
chelating or imaging agents.39−43
Compared to the challenging syntheses of bifunctional
macrocycles like p-SCN-Bn-DOTA, this streamlined route to
p-SCN-Bn-H4octapa should be of significant utility. The bulky
nature of the nosyl protecting groups is admittedly atom
inefficient; however, their use results in a reduced number of
purification steps (compounds 1, 2, and 8 can be purified via
crystallization), greater yields, and, most relevantly, the
accessibility of p-SCN-Bn-H4octapa. The final three steps in
the synthesis of p-SCN-Bn-H4octapa are performed sequen-
tially without purification due to the high polarity and difficult
separation of the intermediates, and the final molecule is
purified via reverse-phase HPLC chromatography.
Following synthesis, the HCl salt of pure H4octapa was
successfully metalated via incubation with indium perchlorate33
[In(ClO4)3] and lutetium chloride [LuCl3] to form quantita-
tively coordinated complexes. Further, all of the new chelators
and their metal complexes were fully chemically characterized
1
using H NMR, 13C NMR, HR-ESI-MS, and ATR-IR where
appropriate (Figures S1−13). Particularly interesting, however,
1
were the H NMR spectra of the metalated H4octapa ligand.
Variable-temperature NMR experiments with In3+ complexes of
DOTA have demonstrated fluxional isomerization in solution,
despite the macrocyclic nature of the chelator.19,33,44 In this
case, however, the 1H NMR spectra obtained for [In(octapa)]−
and [Lu(octapa)]− (6) reveal sharp and distinct coupling
patterns, indicating little fluxional isomerization in solution at
ambient temperature (Figure S5).33
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Figure 1. In silico DFT structure predictions of (a) 8-coordinate structure of [Lu(octapa)]− and (b), 9-coordinate structure of [Lu(octapa)(H2O)]−
as well as the MEP polar-surface area maps (bottom) predicting the charge distribution over the solvent-exposed surface of the metal complexes (red
= negative, blue = positive, representing a maximum potential of 0.254 au and a minimum of −0.254 au, mapped onto electron density isosurfaces of
0.002 Å−3), demonstrating very little difference between the 8- and 9-coordinate solution structures in terms of both geometry and charge
distribution, suggesting little change in physical properties between the 8- and 9-coordinate geometries.
Thermodynamic Stability and Density Functional
Theory Structure Prediction. The thermodynamic stability
constants (log KML) for DOTA with Lu3+ have previously been
determined by spectrophotometric methods (UV) to be 23.5
and ∼2545,46 and by a competitive potentiometric titration with
oxalate to be 29.2 0.2.47 In these studies the only species for
which log KML has been documented is for the ML species (no
metal hydroxide), and titrations were not done in the presence
of sodium, which is known to lower formation constants values
(our titrations are done in 0.15 M sodium for physiologically
relevant conditions).48 These factors, combined with the
inconsistencies in the reported log KML values and the methods
used, prompted us to perform competitive potentiometric
titration experiments of DOTA with EDTA and Lu3+ in order
to provide a formation constant that was determined under
identical experimental conditions to our own system. Using
potentiometric titrations, we have experimentally determined
the thermodynamic stability constant (log KML) for Lu3+ with
H4octapa to be 20.08 0.09 (pM = 19.8) and with DOTA to
be 21.62 0.10 (pM = 17.1). In addition, the log KML value for
Lu3+ with DTPA has been reported in the literature to be 22.4,
and we have calculated a pM value of 19.1.17 Although the
presence of sodium in our experiments can be expected to
lower the stability constant of DOTA with Lu3+ from previously
reported values, and the different experimental methods used
are expected to vary in their results, the low log KML value we
have obtained could be inaccurate as a result of the slow
complex formation kinetics of DOTA. During the potentio-
metric titrations we performed, up to 15 min was allowed for
stabilization of the electrode reading between each addition of
base, which may have been too brief for a proper equilibrium to
be established with DOTA. Previous work has waited over 2
weeks for equilibrium to be reached in this system, which
highlights the sluggish formation kinetics.45 Because of this
uncertainty, our experimentally determined log KML value for
DOTA with Lu3+ cannot be relied on solely or be considered to
be more accurate than previously determined values, and
therefore all reported formation constants must be considered.
The thermodynamic stability constants of the In3+ complexes of
H4octapa, DOTA, and DTPA have been determined to be 26.6
(pM = 26.5), 23.9 (pM = 18.8), and 29.0 (pM = 25.7),
respectively.33 It is important to note that thermodynamic
stability is generally regarded as an unreliable predictor of in
vivo stability on its own,16 and the kinetic inertness of a
radiometal-complex is a much more valuable factor. For
example, the [111In(DOTA)]− complex is widely established
as being significantly more stable than [111In(DTPA)]2− both in
vivo and in vitro, yet the thermodynamic stability constant for
In3+ with DTPA is ∼5 orders of magnitude higher than with
DOTA (29.0 and 23.9, respectively). In fact, pM values, which
are calculated from log KML values while taking into account
ligand pKa’s and metal ion hydroxide formation under specific
and physiologically relevant conditions, are generally consid-
ered more useful in predicting in vivo stability than log KML
values. H4octapa has been shown to have high pM values with
both In3+ (26.5) and Lu3+ (19.8), further substantiating its
promising properties with these metals and their radioactive
isotopologues.
The coordination geometry of both the 8- and 9-coordinate
[Lu(octapa)]− and [Lu(octapa)(H2O)]− complexes were
estimated in silico using density functional theory (DFT)
calculations, and MEP polar surface area maps were super-
imposed on the structure (Figure 1). Both Lu3+ complexes were
found to be highly symmetrical and very similar to one another;
indeed, that the addition of an H2O ligand results in very little
change in metal−ligand bond lengths and angles suggests that
water binding bears little influence on the coordination of the
ligand. Importantly, the [Lu(octapa)]− and [Lu(octapa)-
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Table 1. Chemical and in Vitro Biological Characterization Data for 111In-/177Lu-octapa- and 111In-/177Lu-DOTA-Trastuzumab
Radioimmunoconjugates
radiolabeling conditions and
yield
chelates/
a
specific activity
(mCi/mg)
immunoreactive fraction
serum stability 120 h
b
c
immunoconjugate
isotope
mAb
(%)
(%)
H4octapa-
111ln
177Lu
111ln
15 min, 25 °C, 94%
15 min, 25 °C, 95%
60 min, 37 °C, 50−85%
3.0 0.1
3.0 0.1
3.4 0.1
3.4 0.1
4.0 0.3
3.5 0.4
2.0 0.2
3.4 0.3
99.9 0.02
99.2 0.8
93.2 0.5
95.2 0.2
94.9 0.6
92.4 0.6
91.1 0.6
98.6 0.6
trastuzumab
DOTA-trastuzumab
177Lu
60 min, 37 °C, 50−88%
a
b
c
Isotopic dilution assays, n = 3. Determined immediately prior to in vivo experimentation, n = 6. Calculated for incubation in human serum at 37
°C for 120 h, n = 3.
(H2O)]− DFT structures shown here are quite similar to the
DFT structure of the 8-coordinate [In(octapa)]− complex.33
Taken together, the high log KML and pM values, the absence of
fluxional behavior in solution, and the structural similarity
between the [Lu(octapa)]− and [In(octapa)]− complexes
strongly suggest that these two complexes should exhibit
similar behavior in vitro and in vivo. This is especially important
when considering 111In as a SPECT imaging surrogate isotope
for 177Lu-based radiotherapies.
Bioconjugation and In Vitro Characterization. Syn-
thesis and characterization data aside, the true value of any
radiometal chelator ultimately lies in its behavior in vitro and in
vivo. As a result, the next step in our investigation was to assess
the in vitro and in vivo performance of H4octapa in a model
system based on the HER2/neu-targeting antibody trastuzumab
and HER2-expressing SKOV-3 ovarian cancer cells. In order to
provide a basis for comparison, radioimmunoconjugates
bearing the ubiquitous chelator DOTA were also constructed
and employed in parallel in all in vitro and in vivo experiments.
To begin, purified trastuzumab was incubated under slightly
basic conditions (pH 8.5−9.0) with 4 equivalents of p-SCN-Bn-
H4octapa or p-SCN-Bn-DOTA and purified via size exclusion
chromatography (PD-10, GE Healthcare, U.K.). Subsequent
radiometric isotopic dilution experiments indicated that these
modifications yielded 3.03 0.1 chelates per antibody in the
lower but statistically different values determined for 111In- and
177Lu-DOTA-trastuzumab: 93.2
0.5% and 95.2 0.2%,
respectively (p-values of 0.003 and 0.02, respectively) (Table
1). In order to assay the stability of the radioimmunoconjugates
under biological conditions, all four constructs were incubated
in human serum at 37 °C for a period of 5 days. Over the
course of the experiment, the stability of the 111In-octapa- and
111In-DOTA-trastuzumab was determined to be 94.9
0.6%
and 91.1 0.6%, respectively, and that of the 177Lu-octapa- and
177Lu-DOTA-based conjugates was found to be 92.4
0.6%
and 98.6 0.6%, respectively (Table 1). In the future, acid
dissociation experiments and studies probing the ability of
H4octapa to radiolabel with 111In and 177Lu in the presence of
an excess of metal ions, such as Ca2+, Mg2+, Cu2+, Zn2+, and
Fe3+, are of interest.49,50
Acute Biodistribution Studies. Biodistribution experi-
ments were performed in order to directly compare the in vivo
behavior and pharmacokinetics of the 111In- and 177Lu-based
trastuzumab radioimmunoconjugates. To this end, each of the
four radiolabeled antibodies: 111In-octapa-trastuzumab, 177Lu-
octapa-trastuzumab, 111In-DOTA-trastuzumab, and 177Lu-
DOTA-trastuzumab, were injected via tail vein into female
nude athymic mice bearing subcutaneous SKOV-3 ovarian
cancer xenografts in the right shoulder (∼30 μCi, ∼8−15 μg, in
200 μL of sterile saline; tumor volume ∼100−150 mm3). After
24, 48, 72, 96, or 120 h (n = 4 per time point) the mice were
euthanized via CO2 (g) asphyxiation, and 13 organs, including
the SKOV-3 tumors, were removed, weighed, and assayed for
radioactivity on a γ-counter.
In all four cases, the biodistribution results showed all the
hallmarks of tumor-targeted radioimmunoconjugates, specifi-
cally high levels of uptake in the blood pool at early time points
giving way over time to increasing levels of uptake in the tumor
and, ultimately, very high tumor-to-background organ activity
ratios. In all four cases, the level of uptake in nontarget organs is
roughly similar, with the highest levels of background uptake in
the liver, spleen, and kidneys (Tables S1−3). However, past 24
h, the amount of activity in nontarget organs rarely exceeds 5−
10 %ID/g; for example, at 96 h postinjection, 177Lu-octapa-
trastuzumab displays uptakes of 9.8 1.7 %ID/g, 9.2 1.1 %
ID/g, and 3.8 1.8 %ID/g in the liver, spleen, and kidneys,
respectively. A close inspection of Table 2 reveals not only
slightly higher uptake of 111In- and 177Lu-octapa-trastuzumab in
the bone when compared to the DOTA-trastuzumab
conjugates but also slightly lower uptake of 111In- and 177Lu-
octapa-trastuzumab in the kidneys.
case of p-SCN-Bn-H4octapa and 3.40
0.1 chelates per
antibody in the case of p-SCN-Bn-DOTA. H4octapa-
trastuzumab was then radiolabeled with either 111In or 177Lu
in NH4OAc buffer (pH 5.5, 200 mM) for 15 min at RT, rapidly
producing quantitatively labeled radioimmunoconjugates
(>95% radiochemical yield) in high radiochemical purity
(>99% in each case) and specific activity (4.0 0.3 and 3.5
0.4 mCi/mg, respectively) (Figures S14−15). It is important
to note that these labile kinetics offer a significant improvement
over DOTA-trastuzumab, which required the less favorable
incubation conditions of 1 h at 37 °C to achieve highly variable
radiochemical yields of ∼50−88% and to produce 111In- and
177Lu-labeled radioimmunoconjugates with specific activities of
2.0 0.2 and 3.4 0.3 mCi/mg, respectively (Table 1). These
results are of practical significance because antibodies and
radiometals are very expensive, and so the inconsistent yields
provided when radiolabeling DOTA-trastuzumab could waste
as much as 50% of the antibody construct and radiometal,
greatly increasing cost and making translation to a clinical
radiopharmacy setting more challenging.
The mild radiolabeling conditions afforded by H4octapa-
trastuzumab likely contributed to the extremely high
immunoreactivity of the immunoconjugates as determined by
in vitro cellular assays using SKOV-3 cancer cells: 99.9 0.02%
for 111In-octapa-trastuzumab and 98.7 0.8% for 177Lu-octapa-
trastuzumab. These values compared favorably to the slightly
Far more interesting are the levels of tumor uptake, which
are markedly higher with the 111In-octapa- and 177Lu-octapa-
based variants compared to their 111In-DOTA- and 177Lu-
DOTA-based analogs. For example, at 96 h postinjection the
tumor uptake for 111In-octapa- and 177Lu-octapa-trastuzumab
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Figure 2. SPECT/CT imaging of the 111In/177Lu-octapa- and 111In/177Lu-DOTA-trastuzumab immunoconjugates. Imaging studies in female nude
athymic mice with subcutaneous SKOV-3 xenografts (identified by arrow at right shoulder, tumor volume ∼100−150 mm3), showing transverse
(top) and coronal (bottom) planar images bisecting the tumor, imaged at 24, 48, 72, 96, and 120 h post injection, see Figures S17−20 for 48 and 96
h time points.
was found to be 68.7 20.5 %ID/g and 70.4 25.8 %ID/g,
respectively, compared to 32.8 7.3 %ID/g and 37.0 9.5 %
ID/g for the 111In-DOTA- and 177Lu-DOTA-trastuzumab
constructs. Notably, despite the large range in errors, the p-
values for these differences are 0.04 for the 111In data sets and
0.05 for 177Lu data sets, indicating statistically significant
differences (two-tailed students t test, Tables S1−3). Statistical
analysis of tumor uptake by one-way ANOVA provided p-
values of 0.02 for the 111In data sets and 0.05 for 177Lu data sets.
In imaging, tumor-to-tissue activity ratios are arguably even
more important measures than absolute uptake values, as these
ratios provide quantitative information regarding the contrast
that will be observed during imaging. As a result of the excellent
tumor uptake of the H4octapa-based radioimmunoconjugates,
the tumor-to-background activity ratios were higher than the
corresponding DOTA-based agents for almost all organs. For
example, the tumor-to-blood ratios for 111In-octapa- and 111In-
DOTA-trastuzumab at 120 h were found to be 6.4 3.8 and
cause such a change in vivo. Alternatively, it could be postulated
that while the antigen-binding region of the antibody was only
slightly adversely affected by temperature in the DOTA-
trastuzumab conjugate, a separate region of the immunoglobu-
lin may have suffered bond cleavage or protein structure
disruption during radiolabeling, which in turn affected tumor
uptake and retention. Finally, the differences in pharmacoki-
netic properties from attaching three to four relatively small
chelating moieties to a 150 kD antibody are almost certainly
insignificant, but it is not inconceivable that this modification
could somehow affect the distribution of the radioimmuno-
conjugates in vivo. Experiments are currently underway to
probe the origin of this elevated tumor uptake. Ultimately,
regardless of the causation, the superior tumor uptake and
tumor-to-tissue activity ratios plainly show that 111In-octapa-
and 177Lu-octapa-trastuzumabs are highly effective and versatile
radioimmunoconjugates and, more generally, that H4octapa is a
very promising ligand for the construction of biomolecular
radiopharmaceuticals.
2.2
0.4, respectively, while for 177Lu-octapa- and 177Lu-
DOTA-trastuzumab they were 7.5
respectively (Table S1).
5.8 and 2.5
0.7,
Small Animal SPECT/CT and Cerenkov Luminescence
Imaging. Single photon emission computed tomography
(SPECT) was used in conjunction with standard helical X-ray
CT for in vivo imaging of all 111In- and 177Lu-labeled
immunoconjugates over 5 days, visually demonstrating high
uptake and clear delineation of the SKOV-3 ovarian cancer
xenografts by 111In- and 177Lu-octapa-trastuzumab as well as
The origin of the elevated tumor uptake values observed for
the H4octapa-trastuzumab agents could lie in the enhanced
immunoreactivity conferred by the fast and mild radiolabeling
conditions. However, the difference in immunoreactivity was
only about 5%, a discrepancy that would not be expected to
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111In- and 177Lu-DOTA-trastuzumab (Figure 2). Images taken
at early time points show residual activity in the blood pool at
24 h; however, at later time points, the background activity in
the blood clears and is accompanied by a concomitant increase
in the amount of activity in the tumor, culminating at a point at
which the tumor is by far the most prominent feature in the
image (Figures 2, S17−20). Overall, the image quality, tumor
uptake, and contrast observed are very similar between the
H4octapa- and DOTA-based constructs. In all four cases, high
levels of tumor uptake and excellent tumor-to-background
contrast are observed at later time points, data which correlate
well with the high tumor uptake and tumor-to-tissue ratios
revealed in the biodistribution experiments (Tables 2, S1−3). It
is important to note that while the 111In- and 177Lu-octapa-
trastuzumab images do not appear qualitatively superior to
those produced by the 111In-DOTA- and 177Lu-DOTA variants,
the enhanced tumor-to-background activity ratios of the 111In-
and 177Lu-octapa-based constructs could potentially be of value
in identifying small lesions or metastases in which absolute
uptake of radioactivity is limited and for which contrast is
crucial. The use of Cerenkov radiation (CR) for Cerenkov
luminescence imaging (CLI) has been emerging as a new
imaging modality, with applications such as intraoperative
radionuclide-guided surgery for the resection of cancers.51 In
order to probe the potential of our 177Lu-labeled immuno-
conjugate, we have successfully imaged 177Lu-octapa-trastuzu-
mab via external optical imaging through the skin 24 h post
injection via CLI (Figure 3). To our knowledge, we report the
first published example of in vivo CLI imaging of 177Lu (Figure
3).
streamline radiopharmaceutical production, and aid in the
retention of antibody integrity and immunoreactivity during
radiolabeling. Trastuzumab is a robust antibody and can
generally withstand the radiolabeling conditions required by
DOTA, and so although H4octapa provides significant and
tangible benefits over DOTA as outlined in this manuscript, its
use is not strictly required; however, when working with
antibodies that are more sensitive than trastuzumab and are less
forgiving of elevated temperatures and extended reaction times,
a chelator with more facile reaction kinetics like p-SCN-Bn-
H4octapa may become crucial. The high and reproducible
radiochemical yields that H4octapa provides (>95%) are
additionally important because antibodies are the most
expensive component of these radiolabeled immunoconjugate
systems by a large margin. The inconsistent yields obtained
with DOTA (50−90%) can waste as much as 50% of these
expensive compounds and radiometals and could make it
difficult to reliably produce specific doses in a clinical
radiopharmacy setting. Yet the advantages provided by
H4octapa do not end with facile radiolabeling protocols.
Potentiometric titrations, spectroscopic measurements, and
prolonged serum stability assays indicate that this broadly
versatile chelator forms highly thermodynamically stable and
kinetically inert coordination complexes with isotopes of both
Lu3+ and In3+. Finally, and most importantly, acute biodis-
tribution studies have shown that 111In- and 177Lu-octapa-
trastuzumab specifically and selectively accumulate in SKOV-3
ovarian cancer xenografts in vivo to a degree unmatched by
analogous 111In- and 177Lu-DOTA-trastuzumab radioimmuno-
conjugates. Further, SPECT/CT imaging reveals that both
111In- and 177Lu-octapa-trastuzumab are capable of delineating
HER2-positive tumors in vivo with excellent contrast and high
tumor-to-background activity ratios, producing images that are
comparable to those created using 111In- and 177Lu-DOTA-
trastuzumab. Taken together, these data clearly indicate that p-
SCN-Bn-H4octapa is a versatile and exceedingly promising
bifunctional chelator for the construction of highly potent and
effective 111In- and 177Lu-labeled radiopharmaceuticals.
CONCLUSIONS
■
Ultimately, the major benefit of the acyclic H4octapa chelator
system over macrocyclic chelators, such as DOTA, lies in the
ability to radiolabel with rapid kinetics at RT, a trait which has
the potential to shorten radiolabeling times (<15 min),
EXPERIMENTAL SECTION
■
Materials and Methods. All solvents and reagents were
purchased from commercial suppliers (Sigma Aldrich, St. Louis,
MO; TCI America, Portland, OR; Fisher Scientific, Waltham,
MA) and were used as received unless otherwise indicated.
DMSO used for chelator stock solutions was of molecular
biology grade (>99.9%: Sigma, D8418). 1,4,7,10-Tetraazacy-
clododecane-1,4,7,10-tetraacetic acid para-benzylisothiocyanate
(p-SCN-Bn-DOTA) was purchased from Macrocyclics, Inc.
(Dallas, TX). Methyl-6-bromomethylpicolinate was synthesized
according to a literature protocol.33 Water used was ultrapure
(18.2 MΩ cm−1 at 25 °C, Milli-Q, Millipore, Billerica, MA).
The analytical thin-layer chromatography (TLC) plates were
aluminum-backed ultrapure silica gel (Siliaplate, 60 Å pore size,
250 μM plate thickness, Silicycle, Quebec, QC). Flash column
silica gel was provided by Silicycle (Siliaflash Irregular Silica
Gels F60, 60 Å pore size, 40−63 mm particle size, Silicycle,
Quebec, QC). Automated column chromatography was
performed using a Teledyne Isco (Lincoln, NE) CombiFlash
Rf automated system with solid load cartridges packed with
flash column silica gel and RediSep Rf gold reusable normal-
phase silica columns and neutral alumina columns (Teledyne
Isco, Lincoln, NE). 1H and 13C NMR spectra were recorded on
Bruker AV300, AV400, or AV600 instruments; all spectra were
Figure 3. Cerenkov luminescence image of 177Lu-octapa-trastuzumab.
CLI obtained 24 h postinjection of a female nude athymic mouse
bearing an SKOV-3 ovarian cancer xenograft on the right shoulder,
indicated by white arrow (2 min acquisition time). The bright spots on
the head of the mouse are the result of fluorescent molecules in the
mouse food.
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internally referenced to residual solvent peaks except for 13C
NMR spectra in D2O, which were externally referenced to a
sample of CH3OH/D2O. Low-resolution mass spectrometry
was performed using a Waters liquid chromatography mass
spectrometer (LC-MS) consisting of a Waters ZQ quadrupole
spectrometer equipped with an ESCI electrospray/chemical
ionization ion source and a Waters 2695 HPLC system
(Waters, Milford, MA). High-resolution electrospray-ionization
mass spectrometry (ESI-MS) was performed on a Waters
Micromass LCT time-of-flight instrument. Microanalyses for C,
H, and N were performed on a Carlo Erba EA 1108 elemental
analyzer. The HPLC system used for purification of non-
radioactive compounds consisted of a semipreparative reverse
phase C18 Phenomenex synergi hydro-RP (80 Å pore size, 250
× 21.2 mm, Phenomenex, Torrance, CA) column connected to
a Waters 600 controller, a Waters 2487 dual wavelength
absorbance detector, and a Waters delta 600 pump. 177Lu-
(chelate) analysis was performed using an HPLC system
comprised of a Shimadzu SPD-20A prominence UV−vis, LC-
20AB prominence LC, a Bioscan flow-count radiation detector,
and a C18 reverse phase column (Phenomenex Luna Analytical
250 × 4.6 mm). UV−vis measurements for determining
antibody stock solution concentrations were taken on a
Thermo Scientific Nanodrop 2000 spectrophotometer (Wil-
mington, DE).
serial passage. All animals used were female nude athymic mice
(Taconic Farms, Inc., Hudson, NY).
N,N′-(2-Nitrobenzenesulfonamide)-1,2-diaminoethane
(1). Ethylenediamine (548 μL, 8.2 mmol) was dissolved in THF
(10 mL) and placed in an ice bath, then sodium bicarbonate
(∼2 g) was added, followed by slow addition of 2-nitro-
benzenesulfonyl chloride (4.00 g, 18.1 mmol). The reaction
mixture was allowed to warm to ambient temperature and
stirred overnight. The yellow mixture was filtered to remove
sodium bicarbonate, rotovapped to red oil, and then dissolved
in a minimum volume of dichloromethane and placed in the
freezer overnight. Product precipitated quickly and was filtered
and then washed with cold dichloromethane (3 × 10 mL), and
this process was repeated with the filtrate once more to recover
more material. The faint yellow powder (1) was dried in vacuo
1
for a yield of 87% (∼3.07 g). H NMR (300 MHz, DMSO-d6,
25 °C) δ: 8.16 (br s, 2H), 8.00−7.94 (m, 4H), 7.90−7.83 (m,
4H), 3.00 (s, 4H). 13C NMR (75 MHz, DMSO-d6, 25 °C) δ:
147.62, 134.18, 132.76, 132.41, 129.45, 124.54, 42.39. HR-ESI-
MS calcd for [C14H14N4O8S2 + Na]+: 453.0151; found:
453.0154 [M + Na]+, PPM = 0.7.
N,N′-(2-Nitrobenzenesulfonamide)-N,N′-[6-(methoxycar-
bonyl)pyridin-2-yl]methyl]-1,2-diaminoethane (2). To a
solution of 1 (1.17 g, 2.72 mmol) in dimethylformamide (10
mL, dried over molecular sieves 4 Å) was added methyl-6-
bromomethyl picolinate33 (1.38 g, 5.98 mmol) and sodium
carbonate (∼2 g). The faint yellow reaction mixture was stirred
at 50 °C overnight, filtered to remove sodium carbonate, and
concentrated in vacuo. The crude product was purified by silica
chromatography (CombiFlash Rf automated column system; 40
g HP silica; A: dichloromethane, B: methanol, 100% A to 20%
B gradient) to yield the product 2 as white fluffy solid (95%,
111In was cyclotron produced (Advanced Cyclotron Systems,
Model TR30) by proton bombardment through the reactions
111Cd(p,n)111In and was provided by Nordion as 111InCl3 in
0.05 M HCl. 177Lu was procured from Perkin-Elmer (Perkin-
Elmer Life and Analytical Sciences, Wellesley, MA, effective
specific activity of 29.27 Ci/mg) as 177LuCl3 in 0.05 M HCl.
Labeling reactions were monitored using silica-gel impregnated
glass-microfiber instant thin layer chromatography paper
(iTLC-SG, Varian, Lake Forest, CA) and analyzed on a
Bioscan AR-2000 radio-TLC plate reader using Winscan Radio-
TLC software (Bioscan Inc., Washington, DC). All radio-
labeling chemistry was performed with ultrapure water (>18.2
MΩ cm−1 at 25 °C, Milli-Q, Millipore, Billerica, MA) that had
been passed through a 10 cm column of Chelex resin (BioRad
Laboratories, Hercules, CA). Human blood serum (Sigma,
Sera, human, aseptically filled, S7023−100 mL) competition
solutions were agitated at 550 rpm and held at 37 °C using an
Eppendorf Thermomixer, and then 177Lu(chelate) mixtures
were analyzed using GE Healthcare Life Sciences PD-10
desalting columns (GE Healthcare, United Kingdom, MW <
5000 Da filter) that were conditioned by elution of 25 mL
phosphate-buffered saline (PBS) before use. 177Lu/111In-
immunoconjugates were analyzed using iTLC as described
above and purified using PD-10 desalting columns and Corning
50k MW Amicon Ultra centrifugation filters. Radioactivity in
samples was measured using a Capintec CRC-15R dose
calibrator (Capintec, Ramsey, NJ), and for biodistribution
studies a Perkin-Elmer (Waltham, MA) Automated Wizard
Gamma Counter was used for counting organ activities and
creating calibration curves. SPECT/CT imaging was performed
using a four-headed NanoSPECT/CTPLUS camera (Bioscan
Inc., Washington, DC. USA) with a multipinhole focused
collimator and a temperature-controlled animal bed (Minerve
equipment veterinaire). Imaging of CR was performed using a
IVIS 200 (Caliper Life Sciences) optical imager. Human breast
cancer cell line SKOV-3 was obtained from the American Type
Culture Collection (ATCC, Manassas, VA) and was grown by
1
∼1.88 g) (Rf: 0.9, TLC in dichloromethane). H NMR (300
MHz, CDCl3, 25 °C) δ: 8.10−8.08 (m, 2H), 7.93 (dd, J = 7.8,
0.9 Hz, 2H), 7.75 (t, J = 7.8 Hz, 2H), 7.66−7.62 (m, 4H),
7.60−7.57 (m, 2H), 7.51 (dd, J = 7.8, 0.9 Hz, 2H), 4.71 (s,
4H), 3.91 (s, 6H), 3.51 (s, 4H). 13C NMR (75 MHz, CDCl3,
25 °C) δ: 165.07, 156.21, 147.83, 147.41, 137.89, 133.65,
132.34, 131.93, 131.22, 125.64, 124.09, 124.04, 53.52, 52.65,
46.69. HR-ESI-MS calcd for [C30H28N6O12S2 + H]+: 729.1285;
found: 729.1263, [M + H]+, PPM = −3.0.
N,N′-[6-(Methoxycarbonyl)pyridin-2-yl]methyl-1,2-diami-
noethane (3). To a solution of 2 (1.48 g, 2.03 mmol) in
tetrahydrofuran (10 mL) was added thiophenol (457 μL, 4.47
mmol) and potassium carbonate (excess, ∼1 g). The reaction
mixture was stirred at ambient temperature for 72 h, where a
slow color change from colorless to dark yellow occurred. The
reaction mixture was split into two 20 mL falcon tubes, diluted
with additional tetrahydrofuran, and centrifuged for 5 min at
4000 rpm, and then the solvent was decanted. The potassium
carbonate in the falcon tubes was rinsed/centrifuged/decanted
with tetrahydrofuran 3 times and pooled, and the organic
fractions were concentrated to dryness in vacuo. Please note
that the potassium carbonate can be very sticky (varies between
supplier) and can clog gravity and vacuum filters, and so to
prevent product loss the method of centrifugation is
recommended if a sticky texture is observed after reaction.
The resulting crude yellow oil was purified by silica
chromatography (CombiFlash Rf automated column system;
24 g neutral alumina; A: dichloromethane, B: methanol, 100%
A to 30% B gradient) to yield 3 as clear colorless oil (91%,
∼0.66 g). Compound 3 was purified using neutral alumina, as it
demonstrates an abnormally high affinity for silica and requires
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the use of ammonium hydroxide and >20% methanol to be
eluted, resulting in partial methyl-ester deprotection and
dissolving of some silica. H NMR (300 MHz, MeOD, 25
°C) δ: 8.02 (d, J = 7.5 Hz, 2H), 7.94 (t, J = 7.6 Hz, 2H), 7.66
(d, J = 7.5 Hz, 2H), 3.96 (s, 6H), 2.79 (s, 4H). 13C NMR (75
MHz, MeOD, 25 °C) δ: 167.04, 161.45, 148.48, 139.51, 127.61,
124.84, 54.92, 53.37, 50.00, 49.27. HR-ESI-MS calcd for
[C18H22N4O4 + H]+: 359.1719; found: 359.1720, [M + H]+,
PPM = 0.2.
N,N′-[(tert-Butoxycarbonyl)methyl-N,N′-[6-(methoxycar-
bonyl)pyridin-2-yl]methyl]-1,2-diaminoethane (4). To a
solution of 3 (212 mg, 0.593 mmol) in dry acetonitrile (10
mL, distilled over CaH2) was added tert-butylbromoacetate
(202 μL, 1.36 mmol) and sodium carbonate (∼300 mg). The
reaction was stirred at 60 °C overnight. Sodium carbonate was
removed by filtration, and the crude reaction mixture was
concentrated in vacuo. The crude oil was purified by column
chromatography (CombiFlash Rf automated column system; 24
g HP silica; A: dichloromethane, B: methanol, 100% A to 20%
B gradient) to afford the product 4 as light-yellow oil (92%,
∼0.32 g). 1H NMR (300 MHz, CDCl3, 25 °C) δ: 7.95 (dd, J =
7.2, 1.4 Hz, 2H), 7.80−7.70 (m, 4H), 3.97 (s, 4H), 3.95 (s,
6H), 3.28 (s, 4H), 2.78 (s, 4H), 1.40 (s, 18H). 13C NMR (75
MHz, CDCl3, 25 °C) δ: 170.36, 165.77, 160.79, 147.09, 137.29,
126.02, 123.48, 80.91, 60.51, 56.27, 52.75, 52.39, 28.05. HR-
ESI-MS calcd for [C30H42N4O8 + H]+: 587.3081; found:
587.3085, [M + H]+, PPM = 0.7.
126.78, 126.43, 124.64, 124.50, 123.97, 65.71, 64.25, 62.27,
61.01, 60.51, 59.11, 57.63, 55.96. HR-ESI-MS calcd for
[C20H18175LuN4O8 + 2Na]+: 663.0328; found: 663.0318, [M
+ 2Na]+, PPM = −1.5.
1
1-(p-Nitrobenzyl)ethylenediamine (7). Compound 7 was
prepared according to a literature preparation.52 Product was
purified with a modified procedure using column chromatog-
raphy (CombiFlash Rf automated column system; 40 g HP
silica;, A: 95% dichloromethane 5% ammonium hydroxide, B:
95% methanol 5% ammonium hydroxide, 100% A to 30% B
gradient) to afford 7 as brown/amber oil in a cumulative yield
1
of 40% over 3 steps (∼3 g). H NMR (300 MHz, CDCl3, 25
°C) δ: 7.91 (d, J = 8.9 Hz, 2H), 7.18 (d, J = 8.5 Hz, 2H), 2.80−
2.57 (m, 3H), 2.45−2.31 (m, 2H). 13C NMR (75 MHz, CDCl3,
25 °C) δ: 147.13, 147.71, 129.39, 122.83, 54.26, 47.52, 41.36.
HR-ESI-MS calcd. for [C9H13N3O2 + H]+: 196.1086; found:
196.1084 [M + H], PPM = −1.0.
N,N′-(2-Nitrobenzenesulfonamide)-1-(p-nitrobenzyl)-1,2-
diaminoethane (8). Compound 7 (578 mg, 2.96 mmol) was
dissolved in THF (10 mL) and placed in an ice bath, then
sodium bicarbonate (∼2 g) was added, followed by slow
addition of 2-nitrobenzenesulfonyl chloride (1.58 g, 7.1 mmol).
The reaction mixture was heated to 50 °C and stirred
overnight. The yellow/orange mixture was filtered to remove
sodium bicarbonate, rotovapped to orange oil, and then
dissolved in a minimum volume of dichloromethane and
placed in the freezer. Product precipitated quickly, filtered, and
then washed with cold dichloromethane (3 × 10 mL), and this
process was repeated with the filtrate twice more to recover
more product. The faint yellow powder (8) was dried in vacuo
for a yield of 74% (∼1.24 g) (Rf: 0.90, TLC in 10% methanol in
H4octapa, N,N′-(6-carboxy-2-pyridylmethyl)-N,N′-diacetic
acid-1,2-diaminoethane (5). Compound 4 (138 mg, 0.235
mmol) was dissolved in HCl (10 mL, 6 M) and refluxed for 12
h at 140 °C. The reaction mixture was concentrated in vacuo
and then purified via semiprep reverse-phase HPLC (10 mL/
min, gradient: A: 0.1% TFA (trifluoroacetic acid) in deionized
water, B: 0.1% TFA in CH3CN. 0 to 70% B linear gradient 30
min. tR = 12.6 min, broad). Product fractions were pooled,
concentrated in vacuo, dissolved in 6 M HCl, and then
concentrated in vacuo again to remove trifluoroacetic acid. The
HCl salt H4octapa-4HCl-2H2O (5) was obtained as a white
solid (66% yield, ∼0.097 g, using the molecular weight of the
1
dichloromethane). H NMR (300 MHz, acetone-d6, 25 °C) δ:
8.18−8.15 (m, 1H), 7.99−7.94 (m, 2H), 7.79−7.57 (m, 5H),
7.33 (d, J = 8.5 Hz, 2H), 7.04−6.98 (m, 2H), 4.01 (br s, 1H),
3.41−3.38 (m, 2H), 3.26 (dd, J = 3.4, 13.7 Hz, 1H), 2.98 (m,
1H). 13C NMR (75 MHz, acetone-d6, 25 °C) δ: 206.33, 149.17,
148.14, 147.58, 146.74, 135.23, 134.79, 134.26, 133.89, 133.77,
133.57, 131.67, 131.38, 130.94, 126.00, 125.67, 123.90, 57.67,
49.35, 38.20. HR-ESI-MS calcd for [C21H19N5O10S2 + Na]+:
588.0471; found: 588.0465 [M + Na]+, PPM = −1.0.
1
HCl salt as determined by elemental analysis). H NMR (300
MHz, D2O) δ: 8.11−8.01 (m, 4H, pyr-H), 7.72−7.69 (m, 2H,
pyr-H), 4.53 (s, 4H, Pyr-CH2-N), 3.97 (s, 4H, HOOC-CH2-N),
3.57 (s, 4H, ethylene-H). 13C NMR (75 MHz, D2O) δ: 170.33,
165.27, 151.19, 145.09, 142.64, 128.60, 126.12, 57.81, 54.97,
51.22. IR (neat, ATR-IR): ν = 1721 cm−1 (CO), 1636/1618
cm−1 (CC py). HR-ESI-MS calcd for [C20H22N4O8 + H]+:
447.1515; found [M + H]+: 447.1526, PPM = 0.7. Elemental
analysis: calcd % for H4octapa·4HCl·2H2O (C20H22N4O8·
4HCl·2H2O = 628.2839): C 38.23, H 4.81, N 8.92; found: C
37.94, H 4.66, N 8.59.
[Na][Lu(octapa)] (6). Compound 5 (9.8 mg, 0.013 mmol)
was suspended in 0.1 M HCl (1.5 mL) and LuCl3·6H2O (6.1
mg, 0.015 mmol) was added. The pH was adjusted to 4−5
using 0.1 M NaOH, and then the solution was stirred at RT.
After 1 h the product was confirmed via mass spectrometry, and
the solvent was removed in vacuo to yield [Lu(octapa)][Na]
(6). 1H NMR (600 MHz, D2O, 25 °C) δ: 8.28−8.12 (m, 3H),
8.06 (d, J = 8.2 Hz, 2H), 7.81−7.67 (m, 2H), 4.56−4.48 (m,
1.5H), 4.33−4.05 (m, 2H), 3.81−3.74 (m, 1H), 3.51−3.43 (m,
2H) 3.26−3.16 (m, 4H), 3.00−2.98 (m, 0.5H), 2.57−2.55 (m,
0.5H), 2.12 (br s, 0.5H). 13C NMR (150 MHz, D2O, 25 °C) δ:
180.75, 177.07, 173.29, 172.78, 171.72, 158.17, 157.23, 155.65,
154.34, 150.65, 149.89, 149.55, 143.81, 142.96, 142.54, 141.42,
N,N′-(2-Nitrobenzenesulfonamide)-N,N′-[(tert-
butoxycarbonyl)methyl]-1-(p-nitrobenzyl)-1,2-diamino-
ethane (9). To a solution of 8 (224 mg, 0.390 mmol) in
dimethylformamide (5 mL, dried over molecular sieves 4 Å)
was added tert-butylbromoacetate (169.7 mg, 0.87 mmol) and
sodium carbonate (∼400 mg). The yellow reaction mixture was
stirred at 50 °C overnight, filtered to remove sodium carbonate,
and concentrated in vacuo. The crude product was purified by
silica chromatography (CombiFlash Rf automated column
system; 40 g HP silica; A: ethyl acetate, B: petroleum ether,
100% A to 100% B gradient) to yield 9 as white fluffy solid
1
(88%, ∼0.27 g). H NMR (300 MHz, CDCl3, 25 °C) δ: 7.98
(d, J = Hz, 2H), 7.84−7.39 (m, 8H), 7.25 (d, J = Hz, 2H), 4.46
(Br s, 1H), 4.33−3.85 (m, 6 H), 3.53 (m, 1H), 3.39 (m, 1H),
2.91 (m, 1H), 1.40 (s, 18H). 13C NMR (75 MHz, CDCl3, 25
°C) δ: 168.35, 167.64, 148.00, 147.09, 146.31, 144.83, 133.91,
133.61, 132.53, 131.81, 131.72, 131.60, 131.51, 130.65, 129.85,
124.01, 123.01, 82.71, 82.53, 58.48, 52.47, 50.70, 45.37, 35.04,
27.69. HR-ESI-MS calcd. for [C33H39N5O14S2 + Na]+:
816.1833; found: 816.1838, [M + Na]+, PPM = 0.6.
N,N′-[(tert-Butoxycarbonyl)methyl]-1-(p-nitrobenzyl)-1,2-
diaminoethane (10). To a solution of 9 (151 mg, 0.189 mmol)
in tetrahydrofuran (5 mL) was added thiophenol (41 μL, 0.398
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mmol) and potassium carbonate. The reaction mixture was
stirred at ambient temperature for 72 h, where a slow color
change from colorless to dark yellow occurred. The reaction
mixture was split into two 20 mL falcon tubes, diluted with
additional tetrahydrofuran, centrifuged for 5 min at 4000 rpm,
and then the solvent was decanted. The potassium carbonate in
the falcon tubes was rinsed with tetrahydrofuran and
centrifuged 3 times, pooled, and then concentrated to dryness
in vacuo. The resulting crude yellow oil was purified by silica
chromatography (CombiFlash Rf automated column system; 24
g HP silica; A: dichloromethane, B: methanol, 100% A to 20%
B gradient) to yield 10 as clear colorless oil (89%, ∼0.071 g).
1H NMR (300 MHz, CDCl3, 25 °C) δ: 8.12 (d, J = 8.2 Hz,
2H), 7.35 (d, J = 8.2 Hz, 2H), 3.30 (s, 2H), 3.23−3.22 (m,
2H), 2.87−2.84 (m, 3H), 2.62 (m, 1H), 2.40 (m, 1H), 2.12 (Br
s, 2H, -NH), 1.41 (s, 18H). 13C NMR (75 MHz, CDCl3, 25
°C) δ: 171.68, 171.53, 147.07, 146.51, 130.07, 123.46, 81.17,
81.04, 58.28, 51.57, 49.34, 39.16, 28.01. HR-ESI-MS calcd. for
[C21H33N3O6 + H]+: 424.2448; found: 424.2451, [M + H]+,
PPM = 0.7.
by decanting of the organic phase with a pipet to remove excess
thiophosgene, diluted to a volume of 4.5 mL with deionized
water, and injected directly onto a semiprep HPLC column for
purification (A: 0.1% TFA in deionized water, B: 0.1% TFA in
CH3CN, 100% A to 60% B gradient over 40 min). p-SCN-Bn-
H4octapa (12) was found in the largest peak at Rt = 34 min
(broad, Figure S22), lyophilized overnight, and isolated as a
1
fluffy white solid (50% over 3 steps from 11, ∼0.047 g). H
NMR (400 MHz, MeOD, 25 °C) δ: 8.00−7.87 (m, 4H), 7.59−
7.54 (m, 2H), 7.14 (dd, J = 7.7 Hz, 35.4 Hz, 4H), 5.03 (Br m,
1H), 4.66 (Br m, 2H), 4.49−4.45 (Br m, 1H), 4.11 (Br m, 1H),
3.92 (Br m, 2H), 3.66−3.59 (m, 2H), 3.35−3.18 (Br m, 3H),
2.66−2.60 (m, 1H). 13C NMR (100 MHz, MeOD, 25 °C) δ:
173.10, 167.95, 166.02, 165.82, 161.34, 160.97, 159.40, 151.40,
147.74, 147.21, 138.63, 138.54, 137.60, 135.72, 130.30, 129.75,
127.51, 126.61, 125.65, 124.72, 123.94, 60.31, 59.74, 54.20,
54.14, 33.35. HR-ESI-MS calcd for [C28H27N5O8S + H]+:
594.1659; found: 594.1661, [M + H]+, PPM = 0.3.
Solution Thermodynamics. The experimental procedures
and details of the apparatus closely followed those of a previous
study for H2dedpa with Ga3+ and H4octapa for In3+.30,33 As a
result of the strength of the binding of the Lu3+ complexes,
N,N′-[(tert-Butoxycarbonyl)methyl]-N,N′-[(6-methoxycar-
bonyl)pyridin-2-yl)methyl]-1-(p-nitrobenzyl)-1,2-diamino-
ethane (11). To a solution of 10 (71.7 mg, 0.169 mmol) in dry
acetonitrile (5 mL, distilled over CaH2) was added methyl 6-
(bromomethyl)picolinate33 (85.7 mg, 0.372 mmol) and sodium
carbonate (∼200 mg). The reaction was stirred at 60 °C
overnight. Sodium carbonate was removed by filtration, and the
crude reaction mixture was concentrated in vacuo. The crude oil
was purified by column chromatography (CombiFlash Rf
automated column system; 24 g HP silica; A: dichloromethane,
B: methanol, 100% A to 20% B gradient) to afford the product
[
177Lu(octapa)]− and [Lu(DOTA)]−, the complex formation
constants with these ligands could not be determined directly,
and the ligand−ligand competition method using the known
competitor Na2H2EDTA was used. Potentiometric titrations
were performed using a Metrohm Titrando 809 equipped with
a Ross combination pH electrode and a Metrohm Dosino 800.
Data were collected in triplicate using PC Control (Version
6.0.91, Metrohm). The titration apparatus consisted of a water-
jacketed glass vessel maintained at 25.0 ( 0.1 °C, Julabo water
bath). Prior to and during the course of the titration, a blanket
of nitrogen, passed through 10% NaOH to exclude any CO2,
was maintained over the sample solution. Lutetium ion
solutions were prepared by dilution of the appropriate atomic
absorption standard (AAS) solution. The exact amount of acid
present in the lutetium standard was determined by titration of
an equimolar solution of Lu3+ and Na2H2EDTA. The amount
of acid present was determined by Gran’s method.53
Calibration of the electrode was performed prior to each
measurement by titrating a known amount of HCl with 0.1 M
NaOH. Calibration data were analyzed by standard computer
treatment provided within the program MacCalib54 to obtain
the calibration parameters E0. Equilibration times for titrations
were 10 min pKa titrations and 15 min for metal complex
titrations. Ligand and metal concentrations were in the range of
0.75−1.0 mM for potentiometric titrations. The data were
treated by the program Hyperquad2008.55 The proton
1
11 as light-yellow oil (96%, ∼0.117 g). H NMR (300 MHz,
MeOD, 25 °C) δ: 8.18 (d, J = 8.7 Hz, 2H), 8.11−8.02 (m, 3H),
7.95 (t, J = 7.8 Hz, 1H), 7.73−7.72 (m, 1H), 7.48−7.45 (m,
3H), 4.05 (s, 3H, methyl ester), 3.98 (s, 3H, methyl ester), 3.76
(m, 2H), 3.51 (d, J = 17.4 Hz, 1H), 3.38−3.31 (m, 2H), 3.25−
3.20 (m, 2H), 3.16−3.01 (m, 2H), 2.89−2.82 (m, 2H), 2.71−
2.64 (m, 1H), 2.55−2.51 (m, 1H), 1.39 (s, 18H). 13C NMR (75
MHz, MeOD, 25 °C) δ: 174.44, 173.45, 167.07, 166.81, 160.17,
160.06, 149.25, 148.67, 148.51, 148.09, 140.34, 140.13, 131.60,
129.00, 128.57, 125.14, 125.04, 124.84, 83.39, 83.26, 63.82,
61.82, 58.41, 57.05, 53.78, 53.61, 34.10, 28.38. HR-ESI-MS
calcd for [C36H48N5O10 + H]+: 722.3401; found: 722.3390, [M
+ H]+, PPM = −1.5.
p-SCN-Bn-H4octapa, N,N′-[(Carboxylato)methyl]-N,N′-[(6-
carboxylato)pyridin-2-yl)methyl]-1-(p-benzyl-isothiocyana-
to)-1,2-diaminoethane (12). Compound 11 (118 mg, 0.16
mmol) was dissolved in glacial acetic acid (2.5 mL) with
hydrochloric acid (2.5 mL, 3 M), palladium on carbon (20 wt
%), and hydrogen gas (balloon). The reaction was stirred
vigorously at RT for 1 h, then filtered to remove Pd/C and
washed ad libitum with methanol and hydrochloric acid (3 M).
The crude reaction mixture was concentrated in vacuo,
dissolved in 15 mL of hydrochloric acid (6 M), and refluxed
overnight to facilitate deprotection of the methyl and tert-butyl
esters. HR-ESI-MS calcd for [C27H29N5O8 + H]+: 552.2094;
found: 552.2098, [M + H]+, PPM = 0.7. Without purification,
the crude reaction mixture was dissolved in hydrochloric acid
(1 mL, 3 M) and then reacted with thiophosgene (purchased
suspended in chloroform) in ∼0.2 mL of additional chloroform
(15 equiv, 2.45 mmol) overnight at ambient temperature with
vigorous stirring. The reaction mixture was washed with
chloroform (5 × 1 mL) by vigorous biphasic stirring followed
dissociation constant corresponding to hydrolysis of Lu3+
(aq)
ion included in the calculations were taken from Baes and
Mesmer.56 The acid dissociation constants for DOTA and the
KML value for the lutetium−EDTA complex were taken from
Martell and Smith.17 pM values were calculated at physiolog-
ically relevant conditions of pH 7.4, 100 μM ligand, and 10 μM
metal. All values and errors represent the average of at least
three independent experiments.
Molecular Modeling. Calculations were performed using the
Gaussian 0957 and GaussView packages. Molecular geometries
and electron densities were obtained from DFT calculations,
with the B3LYP functional employing the 6-31+G(d,p) basis
set for first- and second-row elements and the Stuttgart−
Dresden effective core potential, SDD for lutetium.58−60
Solvent (water) effects were described through a continuum
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approach by means of the IEF PCM as implemented in G09.
The electrostatic potential was mapped onto the calculated
electron density surface.
In Vitro Immunoreactivity Assay. The immunoreactivity of
the 111In- and 177Lu-octapa-trastuzumab and 111In- and 177Lu-
DOTA-trastuzumab bioconjugates was determined using
specific radioactive cellular-binding assays following procedures
derived from Lindmo et al.64,65 To this end, SKOV-3 cells were
suspended in microcentrifuge tubes at concentrations of 5.0,
4.0, 3.0, 2.5, 2.0, 1.5, and 1.0 × 106 cells/mL in 500 μL PBS
(pH 7.4). Aliquots of either 111In- and 177Lu-octapa-
trastuzumab or 111In- and 177Lu-DOTA-trastuzumab (50 μL
of a stock solution of 10 μCi in 10 mL of 1% bovine serum
albumin in PBS pH 7.4) were added to each tube (n = 6; final
volume: 550 μL), and the samples were incubated on a mixer
for 60 min at RT. The treated cells were then pelleted via
centrifugation (3000 rpm for 5 min), resuspended, and washed
twice with cold PBS before removing the supernatant and
counting the activity associated with the cell pellet. The activity
data were background corrected and compared with the total
number of counts in appropriate control samples. Immunor-
eactive fractions were determined by linear regression analysis
of a plot of (total/bound) activity against (1/[normalized cell
concentration]). No weighting was applied to the data, and
data were obtained as n = 6.
[
177Lu(chelate)] Radiolabeling. For 177Lu experiments, the
chelators H4octapa, DTPA, and DOTA were used. Aliquots of
each chelator stock solution (1 mg/mL) were transferred to
Corning 2.0 mL self-standing microcentrifuge tubes containing
∼1.2 mCi of 177Lu to a ligand concentration of ∼180 μM and
made up to 1 mL with NaOAc buffer (10 mM, pH 5.0).
H4octapa and DTPA were allowed to radiolabel at ambient
temperature for 10 min, and DOTA was radiolabeled for 1 h at
90 °C. Radiometal complexes were then evaluated using radio-
HPLC (linear gradient A: 0.1% TFA in H2O, B: CH3CN, 0−
80% B over 30 min): [177Lu(octapa)]− tR = 5 min, >99%;
[
177Lu(DOTA)]− tR = 6.8 min, >99%, [177Lu(DTPA)]2− tR =
5.2 min, >99%. Radiolabeled chelators were then used for blood
serum stability assays.
Trastuzumab Antibody Modification/Thiourea Bioconju-
gation. Trastuzumab (purchased commercially as Herceptin,
Genentech, San Francisco, CA) was purified using centrifugal
filter units with a 50 000 molecular weight cutoff (Amicon
ultracentrifuge filters, Ultracel-50: regenerated cellulose, Milli-
pore Corp., Billerica, MA) and phosphate buffered saline (PBS,
pH 7.4) to remove α-α-trehalose dihydrate, L-histidine, and
polysorbate 20 additives. After purification, the antibody was
taken up in PBS pH 7.4. Subsequently, 300 μL of antibody
solution (150−250 μM) was combined with 100 μL PBS (pH
8.0), the pH of the resulting solution was adjusted to 8.8−9.0
with 0.1 M Na2CO3, and 4 equiv of the p-SCN-Bn-H4octapa or
p-SCN-Bn-DOTA was added in 10 μL DMSO. The reactions
were incubated at 37 °C for 1 h, followed by centrifugal
filtration to purify the resultant antibody conjugate. The final
modified antibody stock solutions were stored in PBS (pH 7.4)
at 4 °C.
[
177Lu(chelate)] Blood Serum Competition Experiments.
Frozen human blood serum was thawed for 30 min, and 750 μL
aliquots were transferred to 2.0 mL Corning centrifuge vials.
300 μL of each 177Lu(chelator) was transferred to 750 μL of
blood serum, along with 450 μL of PBS to a total volume of 1.5
mL (n = 3 for each chelator). The final 177Lu(chelate)
concentration present in serum was ∼36 μM. Serum
competition samples were then incubated at 37
0.1 °C
with constant agitation (550 rpm) and analyzed via PD-10 size-
exclusion column elution (filters MW < 5000 Da) at 1.5 and 24
h time points and counted using a Capintec CRC-15R dose
calibrator. 500 μL of each serum/177Lu(chelator) competition
solution (n = 3) was removed from the competition vial,
diluted to 2.5 mL with PBS, and counted. The diluted aliquot
of serum competition mixture was loaded onto a conditioned
PD-10 column. The loading volume (2.5 mL) was eluted into
radioactive waste, and then an additional 3.5 mL of PBS was
loaded, collected, and counted in the dose calibrator as the
serum-bound 177Lu (nonchelate bound). Percent stability was
reported as a percentage of 177Lu still chelate-bound and not
associated with serum proteins (MW < 5000 Da).
111In- and 177Lu-octapa/DOTA-Trastuzumab Radiolabel-
ing. Aliquots of H4octapa/DOTA-trastuzumab immunoconju-
gates were transferred to 2 mL microcentrifuge tubes and made
up to 1 mL of ammonium acetate buffer (pH 5.5, 200 mM),
and then aliquots of 177Lu or 111In were added (∼2−3 mCi).
The H4octapa-trastuzumab mixtures were allowed to radiolabel
at RT for 15 min and then analyzed via iTLC with an eluent of
50 mM EDTA (pH 5), and confirmed reproducible RCY values
of >95% were obtained. The DOTA-trastuzumab mixtures were
heated to 37 0.1 °C for 1 h and then analyzed via iTLC, with
RCYs ranging from ∼50−90%. 30 μL of EDTA solution (50
mM, pH 5) was then added to the reaction mixture, and the
resultant radiolabeled immunoconjugates were then purified
using size-exclusion chromatography (Sephadex G-25 M, PD-
10 column, 30 kDa, GE Healthcare; dead volume = 2.5 mL,
eluted with 1 mL fractions of PBS, pH 7.4) and centrifugal
column filtration (Amicon ultra 50k). The radiochemical purity
of the final radiolabeled bioconjugate was assayed by radio-
iTLC and was found to be >99% in all preparations. In the
iTLC experiments, 111In- and 177Lu-octapa/DOTA-trastuzumab
remained at the baseline, while 177Lu3+/111In3+ ions complexed
as [111In/177Lu]-EDTA eluted with or near the solvent front.
Chelate Number Isotopic Dilution Assay. The number of
accessible H4octapa and DOTA chelates conjugated to
trastuzumab was measured by radiometric isotopic dilution
assays following methods similar to those described by
Anderson et al. and Holland et al.9,61−63 All experiments
were performed in triplicate.
111In- and 177Lu-octapa/DOTA-Trastuzumab Blood Serum
Competition Experiments. Frozen human blood serum was
thawed for 30 min, and 300 μL aliquots were transferred to 2.0
mL Corning centrifuge vials. A portion of radiolabeled
immunoconjugate (∼300 μCi) was transferred to the blood
serum (n = 3 for each chelator). Serum competition samples
were then incubated at 37 0.1 °C with gentle agitation (300
rpm) and analyzed via iTLC with an EDTA eluent (50 mM, pH
5.0) (Bioscan AR-2000) at time points of 0, 24, 48, 72, 96, and
120 h.
Cell Culture. Human ovarian cancer cell line SKOV-3 was
obtained from the American Tissue Culture Collection (ATCC,
Bethesda, MD) and maintained in a 1:1 mixture of Dulbecco’s
Modified Eagle medium: F-12 medium, supplemented with
10% heat- inactivated fetal calf serum (Omega Scientific,
Tarzana, Ca), 2.0 mM glutamine, nonessential amino acids, 100
units/mL penicillin, and 100 units/mL streptomycin in a 37 °C
environment containing 5% CO2. Cell lines were harvested and
passaged weekly using a formulation of 0.25% trypsin/0.53 mM
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EDTA in Hank’s buffered salt solution without calcium and
magnesium.
projections, pitch of 1). Based on the helical CT topogram,
SPECT images were obtained over a range of 85 mm. For the
octapa-based radioimmunoconjugates, the mice were adminis-
tered with either ∼550 μCi of 111In-octapa-trasuzumab (specific
activity ∼3.7 μCi/μg) or ∼450 μCi of 177Lu-octapa-
trastuzumab (3.3 μCi/μg) in 200 μL of sterile saline (0.9%
NaCl) via intravenous tail vein injection. For the DOTA-based
radioimmunoconjugates, the mice were administered with
either ∼400 μCi of 111In-DOTA-trasuzumab (specific activity
∼2.0 μCi/μg) or ∼550 μCi of 177Lu-octapa-trastuzumab (3.4
μCi/μg) in 200 μL of sterile saline (0.9% NaCl) via intravenous
tail vein injection. The amount of immunoconjugate injected
per mouse was ∼150−200 μg. Approximately 5 min prior to
SPECT/CT image acquisition, mice were anesthetized via
inhalation of 2% isoflurane/oxygen gas mixture (Baxter
Healthcare, Deerfield, IL) and placed on the scanner bed.
Anesthesia was maintained during imaging using a reduced
1.5% isoflurane/oxygen mixture. Animals were imaged at 24,
48, 72, 96, and 120 h p.i. Mice injected with 177Lu-labeled
immunoconjugates were imaged for 1.5−2 h each, and mice
injected with 111In-labeled immunoconjugates were imaged for
40 min (24 and 48 h time points), 50 min (72 h), and 60 min
(96 and 120 h). Image collection was performed using Nucline
software (V1.02 build 009), and images were processed and
reconstructed using InVivoScope (2.00 patch3, 64 bit) and
HiSPECT (v 1.4.3049) software. To derive the isotope-specific
calibration factors, a mouse-sized phantom filled with 1.5−2
mCi of 111InCl3 or 177LuCl3 was scanned.
Cerenkov Luminescence Imaging (CLI). Optical images of
CR were obtained using an IVIS 200 (Caliper Life Sciences)
optical imaging machine. This system uses a cryo-cooled
charge-coupled device for high-sensitivity detection of low-
intensity sources. Two minute exposures were obtained of
isoflurane anesthetized mice (n = 2) 24 h post injection of
∼450 μCi of 177Lu-octapa-trastuzumab (3.3 μCi/μg) in 200 μL
of sterile saline (0.9% NaCl) via intravenous tail vein injection.
Statistics. All data used was obtained in at least triplicate (n
= 3), and all values were expressed as mean SD. A student’s
two-tailed t test was used to compare all biodistribution data
sets. A p-value ≤0.05 was considered statistically significant. A
one-way ANOVA analysis was also performed on select data
points; a p-value ≤0.05 was considered statistically significant.
SKOV-3 Xenograft Mouse Models. All experiments were
performed under an Institutional Animal Care and Use
Committee-approved protocol, and the experiments followed
institutional guidelines for the proper and humane use of
animals in research. Six- to eight-week-old athymic nu/nu
female mice (NCRNU-M) were obtained from Taconic Farms
Incorporated (Hudson, NY). Animals were housed in
ventilated cages, given food and water ad libitum, and allowed
to acclimatize for ∼1 week prior to treatment. After several
days, SKOV-3 tumors were induced on the right shoulder by a
subcutaneous injection of 1.0 × 106 cells in a 100 μL cell
suspension of a 1:1 mixture of fresh media/BD Matrigel (BD
Biosciences, Bedford, Ma).
111In- and 177Lu-octapa/DOTA-Trastuzumab Biodistribu-
tion Studies. Biodistribution studies were used to evaluate the
5 day stability and tumor uptake of the 111In- and 177Lu-octapa-
trastuzumab preparations and were compared to the same
system using the “gold standard” 111In- and 177Lu-DOTA-
trastuzumab. DOTA- and H4octapa-trastuzumab constructs
were radiolabeled with 111In- and 177Lu under the same
conditions as described above and prepared for biodistribution
and SPECT/CT imaging studies in nude athymic mice (female,
6−8 weeks old) bearing subcutaneous SKOV-3 ovarian cancer
xenografts (HER2-positive, small with tumor volume ∼100−
150 mm3). The radiolabeled immunoconjugates were purified
via PD-10 size exclusion columns, followed by further
purification and concentration via centrifuge filtration (Amicon
Ultra centrifuge filters, Ultracel-50: regenerated cellulose,
Millipore Corp., Billerica, MA). The purified immunoconju-
gates were suspended in sterile saline (0.9% NaCl) to a
concentration and dose of ∼30 μCi (1.1 MBq) in 200 μL per
mouse. The specific activity of 111In-octapa-trastuzumab was
∼3.7 μCi/μg, and for 177Lu-octapa-trastuzumab it was ∼3.3
μCi/μg. The specific activity of 111In-DOTA-trastuzumab was
∼2.0 μCi/μg, and for 177Lu-DOTA-trastuzumab it was ∼3.4
μCi/μg. The amount of immunoconjugate injected per mouse
was ∼10−15 μg. Each mouse was intravenously injected
through the tail vein and euthanized by CO2 (g) asphyxiation at
time points of 24, 48, 72, 96, and 120 h (n = 4 per time point).
Organs collected after sacrifice included blood, tumor, heart,
lungs, liver, spleen, kidneys, large and small intestines, muscle,
bone (femur), and skin (ear). All organs were rinsed in water
after removal and air dried for 5 min. Tissues were weighed and
counted (calibrated for 111In or 177Lu), the counts were
background and decay corrected from the time of injection and
then converted to the percentage of injected dose (%ID) per
gram of organ tissue (%ID/g). The radioactivity counts
measured in each organ were converted to activity (μCi)
using a calibration curve created from known standards of 111In
or 177Lu.
ASSOCIATED CONTENT
■
S
* Supporting Information
Additional SPECT/CT images, selected H/13C NMR spectra,
1
potentiometric titration data, and select FT-ATR-IR spectra of
final synthesized compounds. This information is available free
of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
lewisj2@mskcc.org; adam@triumf.ca; orvig@chem.ubc.ca
■
111In- and 177Lu-octapa/DOTA-Trastuzumab SPECT/CT
Imaging Studies. Mice with SKOV-3 ovarian cancer xenografts
were imaged with 111In (n = 2) and 177Lu (n = 2) labeled
immunoconjugates, using a four-headed NanoSPECT/
CTPLUS camera (Bioscan Inc., Washington, DC. USA) with
a multipinhole focused collimator and a temperature control
animal bed unit (Minerve Equipment Veterinaire). Nine
pinhole apertures with a diameter of 2.5 mm were used on
each head, with a field of view (FOV) of 24 mm. Settings of the
111In energy peaks were 245 and 171 keV, and the 177Lu peaks
were 208, 112, and 56 keV. A CT at 45 kVp was acquired (180
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was presented at the 2013 Society of Nuclear
Medicine and Molecular Imaging (SNMMI) annual meeting,
and the abstract was awarded the Berson-Yalow award. The
authors thank Blesida Punzalan for skillful tail-vein injections,
Dr. Pat Zanzonico and Valerie A. Longo for aid with SPECT/
12719
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Yordanov, A.; Ma, D.; Schlom, J.; Brechbiel, M. W. Nucl. Med. Biol.
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CT calibration, Dr. Daniel L. J. Thorek for help with Cerenkov
optical imaging, and Michael Doran for cell culture work. We
thank Dr. Cara L. Ferreira and Dr. Dennis W. Wester
(Nordion) for radiochemistry mentoring and collaboration
throughout the project, and Gwendolyn A. Bailey for early
synthesis help. We acknowledge Nordion (Canada) and the
Natural Sciences and Engineering Research Council (NSERC)
of Canada for grant support (CR&D), NSERC for CGS-M/
CGS-D fellowships (E.W.P., C.F.R), and the University of
British Columbia for 4YF fellowships (E.W.P., C.F.R). C.O.
acknowledges the Canada Council for the Arts for a Killam
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bury for a Visiting Erskine Fellowship (2013), and the
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as well as Prof. Dr. Peter Comba and his research group in
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1F32CA1440138-01, B.M.Z.) and the DOE (award DE-
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