64Cu-labeled inhibitors of prostate-specific membrane antigen for PET imaging of prostate cancer

Article abstract of DOI:10.1021/jm401921j

Prostate-specific membrane antigen (PSMA) is a well-recognized target for identification and therapy of a variety of cancers. Here we report five ⁶⁴Cu-labeled inhibitors of PSMA, [⁶⁴Cu]3-7, which are based on the lysine-glutamate urea scaffold and utilize a variety of macrocyclic chelators, namely NOTA(3), PCTA(4), Oxo-DO3A(5), CB-TE2A(6), and DOTA(7), in an effort to determine which provides the most suitable pharmacokinetics for in vivo PET imaging. [⁶⁴Cu]3-7 were prepared in high radiochemical yield (60-90%) and purity (>95%). Positron emission tomography (PET) imaging studies of [⁶⁴Cu]3-7 revealed specific accumulation in PSMA-expressing xenografts (PSMA+ PC3 PIP) relative to isogenic control tumor (PSMA- PC3 flu) and background tissue. The favorable kinetics and high image contrast provided by CB-TE2A chelated [⁶⁴Cu]6 suggest it as the most promising among the candidates tested. That could be due to the higher stability of [⁶⁴Cu]CB-TE2A as compared with [⁶⁴Cu]NOTA, [ ⁶⁴Cu]PCTA, [⁶⁴Cu]Oxo-DO3A, and [⁶⁴Cu]DOTA chelates in vivo.

Full text of DOI:10.1021/jm401921j

Article
pubs.acs.org/jmc
Open Access on 02/17/2015
64Cu-Labeled Inhibitors of Prostate-Specific Membrane Antigen for
PET Imaging of Prostate Cancer
Sangeeta Ray Banerjee,*,† Mrudula Pullambhatla,† Catherine A. Foss,† Sridhar Nimmagadda,†
Riccardo Ferdani,‡ Carolyn J. Anderson,‡ Ronnie C. Mease,† and Martin G. Pomper*,†
†Russell H. Morgan Department of Radiology and Radiological Sciences, Johns Hopkins Medical Institutions, 1550 Orleans Street,
Baltimore, Maryland 21287, United States
‡Department of Radiology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 15219, United States
S
* Supporting Information
ABSTRACT: Prostate-specific membrane antigen (PSMA) is
a well-recognized target for identification and therapy of a
variety of cancers. Here we report five 64Cu-labeled inhibitors
of PSMA, [64Cu]3−7, which are based on the lysine−glutamate
urea scaffold and utilize a variety of macrocyclic chelators,
namely NOTA(3), PCTA(4), Oxo-DO3A(5), CB-TE2A(6),
and DOTA(7), in an effort to determine which provides the
most suitable pharmacokinetics for in vivo PET imaging.
[64Cu]3−7 were prepared in high radiochemical yield (60−
90%) and purity (>95%). Positron emission tomography
(PET) imaging studies of [64Cu]3−7 revealed specific
accumulation in PSMA-expressing xenografts (PSMA+ PC3
PIP) relative to isogenic control tumor (PSMA− PC3 flu) and
background tissue. The favorable kinetics and high image contrast provided by CB-TE2A chelated [64Cu]6 suggest it as the most
promising among the candidates tested. That could be due to the higher stability of [64Cu]CB-TE2A as compared with
[64Cu]NOTA, [64Cu]PCTA, [64Cu]Oxo-DO3A, and [64Cu]DOTA chelates in vivo.
search for small-molecule, functionalized affinity agents for
PSMA that have longer retention and superior pharmacoki-
netics properties for imaging and therapeutic applications is
ongoing.
INTRODUCTION
■
The prostate-specific membrane antigen (PSMA) is emerging
as an attractive target for addressing cancer, whether for
diagnosis or therapy, due to its restricted expression within
normal tissue,1 its elevated expression in the epithelium of
prostate tumors, and within the neovasculature of most solid
tumors tested.2 With respect to prostate cancer, elevated
expression of PSMA is associated with metastasis,3 castrate
resistance,4,5 and progression.6 PSMA has also been used to
guide antibody−drug conjugates and nanoparticles to PSMA-
expressing tissues, including for human studies, some of which
do not involve prostate cancer.7−11 Radiohalogenated, urea-
based, low-molecular-weight inhibitors of PSMA have recently
been explored to image expression of PSMA in prostate tumor
xenografts12,13 as well as in clinical studies.14−16 Radiometals,
including 99mTc,17−23 111In,27−29 64Cu,30 86Y,31 and 89Zr,32,33
have also recently been implemented for imaging PSMA, in
part to leverage the longer physical half-life of these nuclides,
which will be necessary for tracking large peptides, aptamers,
minibodies, antibodies, and nanoparticles. To enable targeting
agents to bind with high affinity to PSMA, a spacer of
approximately 20 Å is generally employed between the PSMA-
targeting group and the metal chelator.21 Moreover, we have
shown that the chelating moiety has a significant effect on the
pharmacokinetics of this class of low-molecular-weight PSMA-
based imaging agents when radiolabeled with 99mTc.34 The
64Cu-Labeled molecules are promising imaging agents for
positron emission tomography (PET) due to the favorable
nuclear characteristics of the isotope (t1/2 = 12.7 h, β+ 17.4%,
Emax = 0.656 MeV, β− 39%, Emax = 0.573 MeV) and its
availability in high specific activity.35 The longer physical half-
life of 64Cu compared to 11C (t1/2 = 20 min) and 18F (t1/2 = 110
min) enables imaging at delayed time points, which allows
sufficient time for clearance from background tissues, resulting
in increased image contrast, particularly for targeting agents
that demonstrate long circulation times such as antibodies and
nanoparticles. Moreover, 64Cu-based PET radiotracers have
demonstrated efficacy for radioimmunotherapy36 comparable
to that for the strictly therapeutic radionuclide, 67Cu (t1/2
=
61.5 h, β− 100%, Emax = 0.121 MeV).37 Accordingly, 64Cu could
be used for imaging and therapy concurrently.38 In vivo stability
of 64Cu-labeled targeted biomolecules depends on the stability
of the corresponding Cu(II)-chelated coordination complex
employed.39−41 Numerous bifunctional chelating agents have
been developed to form stable 64Cu(II) complexes based on
Received: December 15, 2013
Published: February 17, 2014
© 2014 American Chemical Society
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Figure 1. Proposed structures of 64Cu-labeled inhibitors of PSMA.
well-developed copper coordination chemistry42 and have been
used to functionalize small molecules,43,44 peptides,45−48
aptamers,49 and antibodies.50,51 Acyclic and macrocyclic
polyamines tend to have limited stability in vivo with respect
to chelation of copper(II).52,53 Complexes of copper based on
macrocyclic polyamino carboxylates, such as copper chelates of
1,4,8,11-tetraazacyclotetradecane-N,N′,N″,N‴-tetraacetic acid
(TETA) and 1,4,7,10-tetraazacyclodoadecane-N,N′,N″,N‴-tet-
raacetic acid (DOTA), have greater kinetic inertness than
acyclic analogues, although this has not eliminated trans-
chelation.53,54 The cross-bridged tetraamine bicyclic polyami-
nocarboxylates, specifically 1,4,8,11-tetraazabicyclo[6.6.2]-
hexadecane-4,11-diacetic acid (CB-TE2A), provide an improve-
ment over the monocyclic counterparts. The superior kinetic
stability of the copper(II) cross-bridged complexes relative to
DOTA and TETA analogues55−57 is due to the rigidity of the
cross-bridge system. Derivatives of the polyaminocarboxylate
NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) are en-
couraging both because of the convenience of radiolabeling
with 64Cu(II) and the lack of subsequent transmetalation in
vivo.46,47,58
for imaging prostate tumor xenografts in vivo. Although these
low-molecular-weight compounds may clear relatively rapidly
from nontarget sites, that is not always the case, and
increasingly, larger PSMA-specific probes with slower kinetics
are sometimes used as diagnostic and/or therapeutic probes for
PSMA, requiring the longer physical half-life of 64Cu.
Optimization of the chelator for radiolabeling with 64Cu is an
important initial step in its implementation for imaging species
that target PSMA. We investigated the in vitro binding affinity,
mouse biodistribution, and prostate tumor xenograft uptake of
five 64Cu-labeled PSMA inhibitors. To do that, we prepared
urea-based PSMA inhibitors that utilize the Lys-Glu urea motif
and conjugated them with five different macrocyclic chelating
agents, NOTA, CB-TE2A, 3,6,9,15-tetraazabicyclo[9.3.1]-pen-
tadeca-1(15),11,13-triene)-3,6,9-triacetic acid (PCTA), oxa-
4,7,1-tetraazacyclododecane-4,7,10-triacetic acid (oxo-
DO3A),59 and DOTA. Each conjugate was labeled with
63/65Cu/64Cu. Imaging and biodistribution studies in NOD/
SCID mice harboring prostate tumor xenografts demonstrated
that both 64Cu-labeled NOTA- and CB-TE2A-conjugated
radiotracers exhibited favorable pharmacokinetics over the
PCTA, oxo-DO3A, and DOTA-conjugated compounds.
Between the NOTA- and CB-TE2A-chelated compounds, the
Here we study the effect of various chelators for labeling
64Cu on PSMA-targeted ureas with respect to pharmacokinetics
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Scheme 1. Synthesis of [64Cu]3−5
Scheme 2. Synthesis of [64Cu]6
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64Cu-labeled CB-TE2A conjugated [64Cu]6B exhibited superior
tumor-to-background ratios and is the most promising agent
from the series.
Radiochemistry. The radiolabeling of new PSMA ligands
with 64Cu is shown in Schemes 1−2. Briefly, for ligands 3−5
and 7, radiolabeling was performed at pH ∼5.5−6 in acetate
buffer at ∼65 °C for 30 min, whereas for ligand 6, radiolabeling
was achieved in a boiling water bath (95 °C) for 1 h at pH
∼7.5−8 in acetate buffer. Radiolabeled products were obtained
in high yield (>60%, without decay correction) and radio-
chemical purity (>95%) as determined by HPLC and radio
thin-layer chromatography (radio-TLC). Two radiolabeled
peaks were observed for [64Cu]4, likely due to the separation
of diastereomers as previously reported for this class of
bifunctional chelators.60 On the other hand, free ligand 6
contained two nearly inseparable isomeric peaks by HPLC.
Therefore, radiolabeling of 6 was performed using the isomeric
mixture to give two HPLC separable products designated
[64Cu]6A and [64Cu]6B, isolated in 45% and 35% yield,
respectively. We assumed that the presence of an asymmetric
center at the Phe residue of the linker moiety adjacent to CB-
TE2A was responsible for producing those two diastereomeric
compounds. All radioligands were obtained in specific activities
ranging from 2.9 to 9.1 GBq/μmol (78−347 mCi/μmol). The
Log Poct/w values for the radioligands were determined by
octanol−water partition and are shown in Table 1. Surprisingly,
RESULTS
■
Chemical and Radiochemical Syntheses. Macrocyclic
Chelator-Conjugated PSMA Inhibitors. We have synthesized
two different types of ligands as shown in Figure 1 using Lys-
Glu urea as the targeting moiety. For ligands 3−5 we used a
Lys-suberate linker21 and conjugated the linker with
commercially available chelating agents, NOTA-Bn-SCN,
PCTA-Bn-SCN, and oxo-DOTA-Bn-SCN via thiourea bond
formation. For those three bifunctional chelators, the macro-
cyclic backbone was functionalized to carry a benzyl isocyanate
for conjugation with an amine function. Copper(II) com-
pounds for 3−5 are predicted to demonstrate distorted
octahedron geometry.41 Because NOTA is a hexadentate
N3O3 chelator, [64Cu(II)]3 is expected to carry a negative
charge. On the other hand, both PCTA (N4O3) and oxo-
DOTA (N3O4) are heptadentate macrocycles and likely form
neutral compounds with one pendant acetate arm after
complexation.35,41 The Lys-suberate linker was extended
using two phenylalanine residues to prepare 6 and 7 containing
CB-TE2A and DOTA, respectively, as chelating agents. The
purpose of attaching those two phenylalanine residues to the
linker of 6 and 7 is mainly to compensate for the lipophilicity of
the phenylthiourea moiety of compounds 3−5.22,24−26 Note
that one of the pendant acetate arms of the CB-TE2A and
DOTA chelators has been modified to attach the PSMA
targeting moiety via amide bond formation to produce 6 and 7,
respectively. [64Cu]CB-TE2A forms a positively charged
radiotracer when conjugated to a biomolecule, whereas
DOTA-conjugated [64Cu]7 is expected to carry one pendant
acetate, providing an overall neutral compound.
Table 1. Radiolabeling Yield, Log Po/w, and PSMA Inhibitory
Activity
yield (%)
Log Poct/w
Ki (nM)
3
2.84
6.23
[64Cu]3
[64Cu]4
[64Cu]5
∼65−70
∼70−90
∼75−85
−1.17
−1.42
−1.56
[
63/65Cu]3
4
2.03
[
63/65Cu]4
10.76
3.10
5
[
63/65Cu]5
5.47
6
0.19
[64Cu]6A
[64Cu]6B
∼40−45
∼30−35
−2.68
−2.31
[
[
63/65Cu]6A
63/65Cu]6B
3.98
Syntheses of PSMA targeting ligands 3, 4, and 5 are shown in
Scheme 1. Compounds 1 and 2 were prepared as previously
reported.28 Compound 2 was conjugated with commercially
available NOTA-Bn-SCN, PCTA-Bn-SCN, and oxo-DOTA-
Bn-SCN in DMSO in the presence of diisopropylethylamine
(DIEA) at 40 °C for 4 h, to give 3, 4, and 5 in ∼30−40% yield,
respectively, after purification by high-performance liquid
chromatography (HPLC). Synthesis of CB-TE2A-conjugated
ligand 6 was performed using standard fluorenylmethoxycar-
bonyl (Fmoc) solid phase peptide synthesis (SPPS), starting
from Fmoc-Lys(Boc)-Wang resin according to Scheme 2. After
two phenylalanine residues were coupled with the resin bound
lysine, CB-TE2A was conjugated at the N-terminal of the
second phenylalanine residue, after which the compound was
cleaved from the resin by a 1:1 mixture of TFA/CH2Cl2 to
produce 8 in moderate yield (∼20%). The free ε-amine of
lysine of 8 was then conjugated with 128 to produce 6. DOTA-
conjugated 7 was prepared according to our previous report.26
NMR and mass spectrometry (MS) were used to confirm the
identity of newly synthesize compounds. Copper (63/65Cu)
complexes were prepared by incubating conjugates 3−7 with an
aqueous solution of Cu(NO3)2 at 95 °C as shown in Schemes
1−2. The mass spectra of the copper-labeled compounds
showed the expected isotope distribution pattern for natural
copper, which is a mixture of 63Cu (69%) and 65Cu (31%). The
stable copper-labeled conjugates were used as authentic
reference material for chromatography to identify the
corresponding 64Cu-labeled compounds.
4.65
7
11.07
13.26
[64Cu]7
∼65−70
ND
[
63/65Cu ]7
[64Cu]6A and [64Cu]6B were found to be more hydrophilic
than [64Cu]3 in spite of possessing two Phe residues on the
linker and only one carboxylate groups on the chelator
backbone.
In Vitro Binding. Ligands and the corresponding stable
metal-labeled compounds demonstrated high binding affinity to
PSMA, with Ki values ranging from 0.19 to 13.26 nM (Table
1). The known, high-affinity PSMA inhibitor ZJ43 was used as
a reference ligand (Supporting Information Figure S1) and
exhibits a Ki of 4.3 nM.61 The low nanomolar affinities
displayed by these five new copper compounds are similar to
the affinities of the majority PSMA-targeted imaging agents we
have synthesized to date.21,28
Small Animal PET-CT Imaging. Whole body PET-CT
images were obtained for [64Cu]3−7 in intact male severe
combined immunodeficient (SCID) mice (Figures 2−4)
bearing PSMA+ PC3 PIP and PSMA− PC3 flu xenografts in
opposite, upper flanks. Pharmacokinetics derived from the
images were used to determine which compound(s) would be
further evaluated through ex vivo biodistribution. Irrespective
of charge and lipophilicity, all radiotracers, [64Cu]3−7, enabled
visualization of PSMA+ PC3 PIP tumor and kidneys (Figure 2).
Renal uptake of the radiotracers is partially due to the route of
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Figure 2. Whole body PET-CT imaging of PSMA+ PC3 PIP and PSMA − PC3 flu tumor-bearing mice with compounds [64Cu]3, [64Cu]4, [64Cu]5,
[64Cu]6B, and [64Cu]7 at 2.5 h postinjection, respectively. Mice were injected with ∼11.1 MBq (∼300 μCi) of radiotracer IV. PIP = PSMA+ PC3
PIP (solid arrow); flu = PSMA− PC3 flu (unfilled arrow); K= kidney; B = bladder; L = left; R = right. All images are decay-corrected and adjusted to
the same maximum value.
excretion of these agents as well as to specific uptake from the
expression of PSMA in mouse proximal renal tubules.62
Although [64Cu]4, [64Cu]5, and [64Cu]7 demonstrated
moderate to high PSMA+ PC3 PIP tumor uptake, they also
showed significant accumulation within liver at 2.5 h post-
injection (Figure 2), which remained high until 6 h (data not
shown). Radioligands [64Cu]5 and [64Cu]7 exhibited signifi-
cantly higher background uptake, probably related to lower
stability of these 64Cu complexes,58 resulting in trans-chelation
of copper to other, endogenous proteins.56
kidneys, similar to the distribution profile observed with [64Cu]
3. Significantly, both CB-TE2A conjugated diastereomers
[64Cu]6A and [64Cu]6B exhibited similar PET imaging profiles
as shown in Figure 4. Both compounds showed low liver uptake
as early as 20 min after the injection. Consequently, clear
delineation of tumor was achieved even at early time points. By
2.5 h postinjection, radioactivity was largely cleared from
kidneys for both isomers, producing clear target-to-background
contrast for these radiotracers. As a further test of binding
specificity, we imaged animals administered [64Cu]6B after
pretreating them with 50 mg/kg of ZJ43 30 min prior to
radiotracer.61 ZJ43 proved capable of blocking binding of
[64Cu]6B (Supporting Information Figure S2), not only within
the tumor but also within the renal cortex, confirming that
uptake observed in these tissues is PSMA-mediated.62
PET-CT images acquired at 20 min, 6 and 28 h postinjection
of [64Cu]3 (Figure 3) (n = 2) showed clear uptake in PSMA+
Biodistribution. On the basis of PET-CT imaging results,
[64Cu]3, [64Cu]6A, and [64Cu]6B were further assessed in a
biodistribution assays using the same isogenic human prostate
cancer PSMA+ PC3 PIP and PSMA− PC3 flu tumor models (n
= 4). Tables 2 and 3 show the pharmacokinetics in percentage
of injected dose per gram of tissue (% ID/g) in selected organs
for [64Cu]3 and [64Cu]6A at 30 min, 1 h, 2 h, and at 4 or 5 h
postinjection, while Table 4 shows the % ID/g at the optimized
time point of 2 h for [64Cu]3, [64Cu]4, [64Cu]6A, and[64Cu]
6B. All compounds exhibited clear PSMA-dependent binding in
PSMA+ PC3 PIP tumor xenografts, with [64Cu]3 demonstrat-
ing high tumor uptake as early as 30 min postinjection (33.78
9.68% ID/g), peaking at 2 h (38.51 5.68% ID/g) (Table 2),
with extended retention of radioactivity up to 4 h postinjection
(20.64 5.06% ID/g). PSMA+ PC3 PIP to PSMA− PC3 flu
tumor uptake ratios were 14.03 3.71 and 23.50 4.65 at 30
min at 4 h, respectively. The distribution within normal organs
and tissues was also favorable, with low blood and normal tissue
uptake and rapid clearance. The highest nonspecific uptake
Figure 3. Whole body PET-CT imaging of PC3 PIP and PC3 flu
tumor bearing mice with [64Cu]3 at 20 min (left), 6 h (middle), 28 h
(right). Abdominal radioactivity is primarily due to uptake within
kidneys and bladder. PIP = PC3 PSMA+ PIP (solid arrow); flu = PC3
PSMA− flu (unfilled arrow); K= kidney; L = left; R = right, B =
bladder. All images are decay-corrected and adjusted to the same
maximum value.
PC3 PIP tumor. At 20 min and 6 h postinjection, the most
visible tissues were PSMA+ PC3 PIP tumor and kidneys, with
some accumulation of radioactivity observed in liver and
urinary bladder. Radioactivity in liver and kidneys cleared
significantly by 28 h.
observed was in the liver (7.37
0.40% ID/g) and spleen
(28.02 5.82% ID/g) at 30 min postinjection. However, those
values decreased to 4.18 1.61% ID/g and 4.72 1.49% ID/g,
respectively, by 4 h. Kidney uptake was expectedly high and
peaked at 195.44 29.82% ID/g at 30 min and decreased to
108.05 20.50% ID/g by 4 h.
Compounds [64Cu]6A and [64Cu]6B exhibited high radio-
tracer concentration both within PSMA+ PC3 PIP tumor and
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Figure 4. Whole body PET-CT imaging of PC3 PIP and PC3 flu tumor bearing mice with [64Cu]6A (top row) and [64Cu]6B (bottom row) at 20
min, 2.5 h, 12 and 22 h postinjection. Abdominal radioactivity is primarily due to uptake within kidneys and bladder. PIP = PC3 PSMA+ PIP (solid
arrow); flu = PC3 PSMA− flu (unfilled arrow); K= kidney; L = left; R = right, B = bladder. All images are decay-corrected and adjusted to the same
maximum value.
a
Table 2. Tissue Biodistribution for [64Cu]3 in Tumor-Bearing Mice
0.5 h
1 h
2 h
4 h
blood
2.37 0.90
1.78 0.41
6.40 0.59
7.37 0.40
28.02 5.82
1.78 0.31
195.44 29.82
0.57 0.32
4.60 1.42
4.51 1.64
5.65 2.77
33.79 9.68
2.40 0.17
14.03 3.71
15.11 5.38
1.12 0.21
1.39 0.24
5.99 0.70
7.50 1.43
27.58 10.05
1.43 0.38
233.39 22.76
0.60 0.14
5.58 1.43
6.01 2.54
2.48 0.42
29.11 3.02
2.02 0.18
14.37 0.43
26.64 5.12
0.82 0.20
1.11 0.13
4.51 0.97
6.44 0.83
12.33 6.32
0.76 0.34
199.69 48.44
0.34 0.12
3.68 0.41
3.80 1.67
13.33 8.35
38.51 5.68
1.92 0.23
19.96 0.81
48.75 10.27
0.45 0.20
0.71 0.26
2.49 0.99
4.18 1.61
4.72 1.49
0.65 0.75
108.05 20.50
0.21 0.10
2.44 1.30
2.42 1.19
1.70 0.97
20.64 5.06
1.04 0.37
23.50 4.65
50.01 15.54
heart
lung
liver
spleen
fat
kidney
muscle
small intestine
large intestine
bladder
PC-3 PIP
PC-3 flu
PIP:flu
PIP:blood
a
Values expressed are in % ID/g standard deviation; n = 4 for all tissues.
Table 3 shows the organ-related % ID/g of uptake for [64Cu]
stomach, and pancreas, showed lower uptake (∼2% ID/g at 1
h) and much faster clearance than for [64Cu]3 and decreased to
below 1% ID/g by 5 h.
6B. Similar to [64Cu]3, [64Cu]6B showed the highest PSMA-
dependent tumor uptake with 29.50
8.10% ID/g at 1 h
postinjection. Tumor uptake remained high, decreasing to
18.69 2.88% ID/g at 5 h. The PSMA+ PC3 PIP to PSMA−
PC3 flu ratios were 19.36 3.98 at 30 min, 79.40 15.83 at 2
h, and 146.79 32.20 at 5 h. The PSMA+ PC3 PIP tumor-to-
muscle ratio reached a maximum value of 436.14 202.71 at 5
h, nearly 3-fold higher than those observed with [64Cu]3. Renal
uptake for [64Cu]6B was highest at 30 min at 256.11 75.72%
ID/g, much higher than that seen for [64Cu]3, however, it
cleared much more rapidly, decreasing to 3.54 0.04% ID/g
by 5h. Nontarget organs, such as blood, heart, liver, spleen,
Because [64Cu]3, [64Cu]4, and [64Cu]6 demonstrated
superior pharmacokinetics to [64Cu]5 and [64Cu]7, a one-
time point (2 h) biodistribution study (Table 4) was performed
with [64Cu]3, [64Cu]4, and [64Cu]6B. Syntheses and
biodistribution studies were performed on the same day, as
close to one another as possible to minimize the changes in
specific activity of the radiotracer. The biodistribution and
imaging data reveal several important points regarding the in
vivo properties of these compounds. The NOTA-chelated
radiotracers [64Cu]3, PCTA-chelated [64Cu]4, and CB-TE2A-
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a
Table 3. Tissue Biodistribution for [64Cu]6B in Tumor-Bearing Mice
0.5 h
1 h
2 h
5 h
blood
2.50 1.08
0.92 0.27
2.70 0.73
0.73 0.17
1.02 0.47
0.90 0.23
5.80 2.61
0.87 0.15
256.11 75.72
0.33 0.01
0.85 0.41
0.88 0.42
3.99 0.26
23.14 2.20
1.29 0.12
19.36 3.98
9.13 5.23
69.35 4.29
0.64 0.24
0.32 0.08
0.83 0.11
0.42 0.04
0.48 0.28
0.31 0.19
2.14 0.58
0.87 1.32
148.60 48.41
1.71 2.29
1.38 1.89
0.77 0.65
7.06 3.75
29.50 8.10
0.66 0.26
48.41 14.25
47.47 7.24
91.30 93.17
0.26 0.13
0.13 0.05
0.45 0.21
0.30 0.06
0.25 0.06
0.13 0.05
0.65 0.15
0.19 0.10
65.39 21.16
0.17 0.05
0.26 0.07
0.30 0.08
2.21 0.57
20.46 2.90
0.26 0.04
79.40 15.83
101.17 6.70
132.99 43.97
0.06 0.05
0.06 0.01
0.15 0.01
0.21 0.03
0.10 0.03
0.05 0.04
0.25 0.07
0.05 0.05
3.54 0.04
0.05 0.02
0.11 0.10
0.14 0.05
1.43 1.41
18.69 2.88
0.13 0.03
heart
lung
liver
stomach
pancreas
spleen
fat
kidney
muscle
small intestine
large intestine
bladder
PC-3 PIP
PC-3 flu
PIP:flu
146.79 32.30
1123.96 25.83
436.14 202.71
PIP:blood
PIP:muscle
a
Values expressed are in % ID/g standard deviation; n = 4 for all tissues.
a
Table 4. Tissue Biodistibistion for [64Cu]3, [64Cu]4, [64Cu]6A, and [64Cu]6B at 2 h Post-Injection in Tumor-Bearing Mice
[64Cu]3
[64Cu]4
[64Cu]6A
[64Cu]6B
blood
1.06 0.29
1.48 0.14
5.31 0.74
8.63 0.92
3.88 0.82
2.22 0.34
13.42 1.18
0.76 0.77
125.40 42.47
0.33 0.08
6.24 1.46
10.13 6.83
12.47 10.30
16.58 3.15
2.12 0.25
11.00 0.73
16.52 5.14
46.98 19.05
1.87 0.63
2.71 0.13
10.16 2.29
17.04 1.44
6.37 0.78
3.51 0.67
9.27 2.28
1.18 0.59
166.57 42.39
0.71 0.32
10.95 3.70
10.10 4.76
10.56 6.20
24.13 10.06
3.88 0.62
6.22 1.86
12.87 1.87
38.34 21.69
0.20 0.03
0.26 0.10
1.17 0.61
1.68 0.38
0.62 0.42
0.46 0.44
0.39 0.30
0.27 0.26
23.55 8.96
0.11 0.05
0.74 0.32
1.49 0.75
7.98 8.32
11.10 5.42
0.36 0.09
30.90 7.92
52.41 17.34
97.31 42.46
0.20 0.07
0.35 0.21
0.89 0.15
1.63 0.72
0.62 0.17
0.80 0.92
0.90 0.23
0.05 0.02
24.87 11.26
0.12 0.05
0.68 0.13
1.28 0.70
5.92 4.94
16.89 5.73
0.40 0.12
41.75 5.54
86.97 15.22
138.88 30.86
heart
lung
liver
stomach
pancreas
spleen
fat
kidney
muscle
small intestine
large intestine
bladder
PC-3 PIP
PC-3 flu
PIP:flu
PIP:blood
PIP:muscle
a
Values expressed are in % ID/g standard deviation; n = 4 for all tissues.
chelated [64Cu]6B showed high uptake in PSMA+ PC3 PIP
tumor, highest for [64Cu]4 (24.13 10.06% ID/g). However,
[64Cu]4 also exhibited slightly higher nonspecific uptake in
PSMA− PC3 flu tumor and other organs, including blood,
muscle, heart, liver, and stomach, resulting in lower tumor-to-
organ ratios than for [64Cu]3 and [64Cu]6B. In the imaging
studies, [64Cu]4 exhibited considerable background radio-
activity primarily due to high liver uptake of [64Cu]4.
Diastereomers [64Cu]6A and [64Cu]6B, each containing the
CB-TE2A chelating agent, possessed similar in vivo properties
and exhibited significantly lower normal tissue uptake,
including kidney, than either [64Cu]3 or [64Cu]4.
compounds for clinical application. It is generally accepted that
the overall biologic profile of radiolabeled ligands is determined
not only by receptor-specific binding but also by nonspecific
interactions. Such off-target effects rely on the overall
physicochemical features of the radiolabeled compound, e.g.,
molecular weight, charge, lipophilicity, and metabolic stability.
The chelator used to attach the radionuclide to the targeting
agent plays a key role in establishing those physicochemical
features, particularly for agents smaller than antibodies and
nanoparticles, namely those of relatively low molecular weight
(<1500 Da). Two key findings emerged from this study. First,
both NOTA and CB2-TE2A-conjugated radiotracers [64Cu]3
and [64Cu]6 demonstrated significantly higher in vivo stability,
as evidenced by their lower liver uptake than the other three
radiotracers, [64Cu]4, [64Cu]5, and [64Cu]7, which utilized
PCTA, oxo-DOTA, and DOTA, respectively. High liver uptake
and slow blood clearance for those latter three radiotracers are
DISCUSSION
■
We have evaluated five 64Cu-labeled PSMA-targeted, urea
conjugates using different chelating agents with the aim of
optimizing in vivo PET imaging properties of this class of
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indicative of free Cu(II), which is sequestered in liver.56,63
Second, liver uptake and blood concentration are much lower
at all time points for [64Cu]6A−B compared to [64Cu]3,
suggesting higher in vivo stability of CB-TE2A-conjugated
[64Cu]6A−B compared to NOTA-conjugated [64Cu]3. That
might also be related to the higher hydrophilicity of [64Cu]6A−
B compared to [64Cu]3 (Log Po/w = −2.68 for [64Cu]6B
compared to −1.17 for [64Cu]3). Low liver uptake of CB-
TE2A-chelated [64Cu]6 ligands might also be related their
higher stability in forming complexes with Cu(II) and Cu(I),
assuming Cu(II) reduction is the primary reason for trans-
chelation, as reported previously.56 It is worth mentioning that
[64Cu]3, which possesses an overall negative charge, exhibited
higher kidney and spleen uptake that was similar to that
observed for 99mTc-oxo labeled PSMA-targeted compounds.34
Both kidney and tumor uptake of [64Cu]6B were specifically
blocked by ZJ43, demonstrating PSMA-mediated renal uptake,
however, much faster renal clearance of [64Cu]6B was observed
compared to clearance from PSMA+ PIP tumor. We and others
have seen a similar pattern of faster clearance of radioactivity
from PSMA-expressing kidney than PSMA+ PIP tumor,
presumably associated with more rapid flow through normally
organized renal parenchymal vasculature compared to the
relatively disorganized vasculature of the xenograft.26,64,65 As an
extension of that particular pharmacokinetic feature, [64Cu]6
exhibited the highest tumor-to-normal tissue ratios in this series
of agents and the ratios increased with time, thereby providing
high image contrast. Moreover, no bone uptake was observed
for [64Cu]6, indicating that this agent may find application for
detection of prostate cancer metastases, which are preferentially
found in bone.
nonlinear nature of the desferrioxamine (DFO) chelating
agent.68 More recently, studies in biopsy tissue from patients
with prostate cancer and cell lines have demonstrated a
correlation between PSMA cell−surface expression and
androgen activity using 64Cu-labeled anti-PSMA antibody
([64Cu]J591).20 ImmunoPET detection of PSMA could
provide a path for quantitative monitoring of successful
androgen blockade in patients. Low-molecular-weight urea-
based agent [64Cu]6 could be an inexpensive alternative for
those applications. Furthermore, results obtained with [64Cu]6
suggest that delayed imaging with 64Cu-labeled PSMA-targeted
ligands using CB-TE2A can improve the visualization of PSMA
+ tumors in vivo anywhere outside of central nervous system
and may allow imaging of tumors with lower PSMA expression
levels than possible with existing 18F- or 68Ga-labeled PSMA-
targeted imaging agents.
CONCLUSION
■
We describe five new low-molecular-weight, urea-based, 64Cu-
labeled, PSMA-targeted radiotracers that incorporated well-
established chelating agents. All compounds demonstrated high
tumor uptake and retention with the choice of chelator having a
profound effect on pharmacokinetics. Our data revealed that
[64Cu]6, which utilizes the CB-TE2A chelator, demonstrated
improved biodistribution with rapid clearance from normal
tissues, including kidney, resulting in significantly improved
image contrast. Accordingly, [64Cu]6 provides a potentially
clinically viable imaging agent for PSMA+ tissues, particularly if
delayed imaging, obtainable with a 64Cu-labeled agent, is
required.
The imaging and biodistribution results demonstrate higher
in vivo stability and renal clearance for CB-TE2A-chelated
[64Cu]6 compared to NOTA-chelated [64Cu]3, which appears
to be in contradistinction to previous reports that involve
similar comparison for agents that target somatostatin receptor
and αvβ3 integrin. In those reports [64Cu]NOTA-conjugated
radiopeptides provided faster renal clearance than those
conjugated with [64Cu]CB-TE2A.46,47 Although unclear at
this time, this difference between 64Cu-labeled species for
targeting of PSMA vs somatostatin receptor or integrin may be
associated with other physical properties unique to each system
such as binding affinity or capacity for internalization of the
cognate ligands. It nevertheless underscores the uniqueness of
each system and indicates that one chelator cannot be assumed
to outperform another in a new, untested system.
There are several classes of imaging agents available for PET-
based molecular imaging of PSMA, including antibodies,4,5,32,66
antibody fragments,67 aptamers,49 and low-molecular-weight
PSMA-binding affinity agents.14,64 They have widely varying
pharmacokinetics, however, each class has shown PSMA-
specific binding in preclinical studies. Rockey and colleagues
have recently optimized conditions for conjugating 64Cu to a
PSMA-targeting aptamer with different chelators, which has
shown PSMA-mediated uptake in PSMA+ 22Rv1 prostate
tumor cells relative to PSMA− PC3 cells.49 The radiolabeled
monoclonal antibodies [64Cu]DOTA-3/A1214 and [89Zr]DFO-
J59155 both showed values of approximately 3:1 at 48 h post-
injection for PSMA+ to PSMA- tumor. Among the long-lived
PET radionuclides, 64Cu and 89Zr, the stability of the
[89Zr]DFO complex is limited as shown in several in vitro
and in vivo in studies, which is reflected in a bone accumulation
ranging from 3 to 15% and assumed to be associated with the
EXPERIMENTAL SECTION
■
Solvents and chemicals purchased from commercial sources were of
analytical grade or better and used without further purification. All 9-
fluorenylmethyloxycarbonyl (Fmoc) protected amino acids including
the Fmoc-Lys(Boc)-Wang resin, 1-hydroxybenzotriazole monohy-
drate, and 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexa-
fluorophosphate (HBTU) were purchased from Chem Impex
International Inc. (Wooddale, IL). [64Cu]CuCl2 was purchased from
the University of Wisconsin. DOTA-tris(t-butyl ester)-monoacid, p-
SCN-Bn-NOTA, p-SCN-Bn-PCTA, and p-SCN-Bn-oxo-DO3A were
received from Macrocyclics Inc. (Dallas, TX). Copper(II) chloride,
triethylsilane (Et3SiH), diisopropylethylamine (DIEA), and triethyl-
amine (TEA) were purchased from Sigma-Aldrich (St. Louis, MO). All
other chemicals were purchased from Thermo Fisher Scientific
(Pittsburgh, PA) unless otherwise specified. Analytical thin-layer
chromatography (TLC) was performed using Aldrich aluminum-
backed 0.2 mm silica gel Z19, 329-1 plates and visualized by ultraviolet
light (254 nm), I2, and 1% ninhydrin in EtOH. Flash chromatography
was performed using silica gel (MP SiliTech 32-63 D 60 Å) purchased
from Bodman (Aston, PA). All in vitro PSMA binding studies and
determination of partition coefficient experiments were performed in
triplicate to ensure reproducibility. 1H NMR spectra were recorded on
a Bruker UltrashieldTM 400 MHz spectrometer. Chemical shifts (δ)
are reported in ppm downfield by reference to proton resonances
resulting from incomplete deuteration of the NMR solvent. Low
resolution ESI mass spectra were obtained on a Bruker Daltonics
Esquire 3000 Plus spectrometer. High resolution mass spectra were
obtained by the University of Notre Dame Mass Spectrometry and
Proteomics Facility, Notre Dame, IN, using ESI either by direct
infusion on a Bruker microTOF-II or by LC elution via an ultrahigh
pressure Dionex RSLC with C18 column coupled with a Bruker
microTOF-Q II.
HPLC purification of compounds 3−7 was performed using a
Phenomenex C18 Luna 10 × 250 mm2 column on a Waters 600E Delta
LC system with a Waters 486 variable wavelength UV/vis detector,
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both controlled by Empower software. HPLC was performed using the
following methods. Method 1: solvent A (0.1% TFA in water) and
solvent B (0.1% TFA in in acetonitrile), flow rate 8 mL/min. The
elution gradient was 90% A and 10% B for 0−5 min and 90% A to 0%
A and 10% B to 100% B over 6−30 min. Methods 2−5 were isocrati,c
with a flow rate 4 mL/min. Method 2: 85% A and 15% B for 0−50
min. Method 3: 82% A and 18% B for 0−40 min. Method 4: 83% A
and 17% B for 0−40 min. Method 5: 78% A and 22% B for 0−30 min.
Method 6: 80% A and 20% B for 0−30 min, flow rate 1 mL/min, and a
Waters Novapak C18 150 × 3.9 mm2 column was used. HPLC
purification of [64Cu]3-7 were performed on a Varian Prostar System
(Palo Alto, CA), equipped with a Varian ProStar 325 UV−vis variable
wavelength detector and a Bioscan Flow-Count in-line radioactivity
detector, all controlled by Galaxie software. The specific radioactivity
was calculated as the radioactivity eluting at the retention time of
product during the preparative HPLC purification divided by the mass
corresponding to the area under the curve of the UV absorption. The
purity of tested compounds as determined by analytical HPLC with
absorbance at 220 nm were >95%.
1; Rt 13.40 min). Yield ∼10 mg (0.076 μmol, ∼23%) after HPLC
1
purification and lyophilization. H NMR (DMSO-d6) δ: 8.12 (m, 1H,
HNCO(Lys-linker)), 7.80−7.75 (m, 1H, HNCO(Lys)), 7.14 (d, 2H,
Bz), 6.79 (d, 2H, Bz), 6.32 (m, 2H, NH(CO)NH), 4.42−3.99 (m, 3H,
HC(NHCO2(Glu), HC(NHCO2(Lys), HC(NHCO2(Lys)), 3.49−
3.31 (m, 11H, NCH2(DO3A), CHCH2(DO3A), OCH2)), 3.21−
3.02 (m, 16H, (CH2)2(DO3A), H2C-Bz, H2CNH(Lys-linker),
H2CNH(Lys)), 2.41−2.08 (m, 6H, H2CCO2(Glu), H2CCO2(linker),
H2CCO2(linker)), 1.91−1.50 (m, 6H, H2CCH(Glu), H2CCH(Lys-
linker), H2CCH(Lys)), 1.50−1.22 (m, 16H, (CH2)2(Lys),
(CH2)2(Lys-Linker), (CH2)4(linker). ESMS m/z: 1098 [M + 1]+,
HRESI+ MS: calcd for C48H76N9O18S, 1098.5029 [M + H]+; found,
1098.5048.
(14S,29S,33S)-4,7-Dibenzyl-1-(11-(carboxymethyl)-1,4,8,11-
tetraazabicyclo[6.6.2]hexadecan-4-yl)-2,5,8,16,23,31-hexaoxo-
3,6,9,15,24,30,32-heptaazapentatriacontane-14,29,33,35-tet-
racarboxylic Acid, Compound 6. Fmoc-Lys(Boc)-Wang resin (100
mg, 0.43 mM) was allowed to swell with CH2Cl2 (3 mL) followed by
DMF (3 mL). A solution of 20% piperidine in DMF (3 × 3 mL) was
added to the resin that was then shaken gently on a mechanical shaker
for 30 min at ambient temperature. The resin was washed with DMF
(3 × 3 mL) and CH2Cl2 (3 × 3 mL). Formation of free amine was
assessed by the Kaiser test. After swelling the resin in DMF, a solution
of Fmoc-Phe-OH (3 equiv), HBTU (3 equiv), HOBt (3 equiv), and
DIPEA (4.0 equiv) in DMF was added and gently shaken for 2 h. The
resin was then washed with DMF (3 × 3 mL) and CH2Cl2 (3 × 3
mL). The coupling efficiency was assessed by the Kaiser test. The
aforementioned sequence was repeated for two more coupling steps
with Fmoc-Phe-OH and CB-TE2A, respectively. The final compound
was cleaved from the resin using TFA:CH2Cl2 (1:1) and concentrated
under vacuum to produce 8. The concentrated product was purified by
using a C18 SepPak Vac 2g column. The product was eluted with a
solution 70/30 water/acetonitrile (0.1% TFA in each). ESI-MS: 635
[M]+. To a solution of 8 (20 mg, 31 μmol in 500 μL of DMSO) (27
mg, 47 μmol) and DIEA (50 μL) was added 1, and the reaction
mixture was left at ambient temperature for 2 h. The solution was
diluted in 10 mL of water and was purified by HPLC (method 1, time
(Rt), 20 min). Yield ∼10 mg (0.076 μmol, ∼22%) after HPLC
Synthesis and Characterization of Compounds 1−7.
Compounds 1,28 and 2,26 and 726 were prepared following our
previous reports. Compounds 3−5 were prepared following a general
procedure as described for compound 3.
9 , 1 6 , 24- T r i o x o -1 -t h i o xo -1 - ((4 -( (( S) -1 , 4 , 7 -t ri s -
(carboxymethyl)-1,4,7-triazonan-2-yl)methyl)phenyl)amino)-
2,8,17,23,25-pentaazaoctacosane-7,22,26,28-tetracarboxylic
Acid, Compound 3. To a solution of 2 (20 mg, 0.33 μmol in 500 μL
of DMSO) was added NOTA-Bn-SCN (18.5 mg, 0.33 μmol in 500 μL
of DMSO) and DIEA (100 μL), and the solution was kept at 40 °C for
4 h. The reaction mixture was then evaporated to dryness, and the
crude residue was dissolved in 2 mL of water and loaded on a
prepacked C18 column (5.5 g, Agilent SF10). The product was eluted
with 80/20−70/30 H2O (0.1%TFA)/CH3CN (0.1% TFA) solution.
The fractions containing the products were combined together and
evaporated to dryness to obtain a colorless solid. The solid thus
obtained was further purified by HPLC (method 1, retention time, Rt
15.80 min). Yield ∼8 mg (0.076 μmol, ∼23%) after HPLC purification
1
and lyphilization. H NMR (DMSO-d6) δ: 8.15 (m, 1H, HNCO(Lys-
linker)), 7.75 (m, 1H, HNCO(Lys)), 7.14 (d, 2H, Bz), 6.78 (d, 2H,
Bz), 6.34 (m, 2H, NH(CO)NH), 4.40−3.99 (m, 3H, HC-
(NHCO2(Glu), HC(NHCO2(Lys), HC(NHCO2(Lys)), 3.40−3.30
(m, 7H, NCH2, CHCH2, (NOTA)), 3.21−3.03(m, 16H,
(CH2)2(NOTA), H2C-Bz, H2CNH(Lys-linker), H2CNH(Lys)),
2.40−2.09 (m, 6H, H2 CCO2 (Glu), H2 CCO2 (linker),
H2CCO2(linker)),1.89−1.55 (m, 6H, H2CCH(Glu), H2CCH(Lys-
linker), H2CCH(Lys)), 1.55−1.21 (m, 16H, (CH2)2(Lys),
(CH2)2(Lys-Linker), (CH2)4(linker)). ESMS m/z: 1054 [M + H]+.
HRESI+ MS: calcd for C46H72N9O17S, 1054.4767 [M + H]+; found,
1054.4771.
9,16,24-Trioxo-1-thioxo-1-((4-(((4S)-3,6,9-tris-
(carboxymethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1-
(14),12-dien-4-yl)methyl)phenyl)amino)-2,8,17,23,25-pentaa-
zaoctacosane-7,22,26,28-tetracarboxylic Acid, Compound 4.
The compound was purified by HPLC (method 1; Rt 15.60 min).
Yield ∼12 mg (0.076 μmol, ∼23%) after HPLC purification and
lyophilized. 1H NMR (DMSO-d6) δ: 8.14 (m, 1H, HNCO(Lys-
linker)), 7.80−7.75 (m, 2H, HNCO(Lys), Py), 7.32 (d, 2H, Py), 7.15
(d, 2H, Bz), 6.78 (d, 2H, Bz), 6.35 (m, 2H, NH(CO)NH), 4.42−3.99
(m, 7H, HC(NHCO2(Glu), HC(NHCO2(Lys), HC(NHCO2(Lys),
H2C-Py)), 3.42−3.31 (m, 7H, CHCH2, NCH2(PCTA)), 3.21−3.02
(m, 12H, (CH2)2(PCTA), H2C-Bz, H2CNH(Lys-linker), H2CNH-
(Lys)), 2.41−2.08 (m, 6H, H2CCO2(Glu), H2CCO2(linker),
H2CCO2(linker)), 1.89−1.55 (m, 6H, H2CCH(Glu), H2CCH(Lys-
linker), H2CCH(Lys)), 1.55−1.21 (m, 16H, (CH2)2 (Lys),
(CH2)2(Lys-Linker), (CH2)4(linker)). ESMS m/z: 1132 [M + H]+.
HRESI+ MS: calcd for C51H75N10O17S, 1131.5032 [M + H]+; found,
1131.5011.
1
purification and lyophilization. H NMR (DMSO-d6): 9.02−8.07 (m,
12H, HNCO(Lys-linker), HNCO(Lys), PhCH2), 6.34 (m, 2H,
NH(CO)NH), 4.62 (m, 2H, NHCO2(Phe)), 4.37−4.28 (m, 3H,
(m, 3H, HC(NHCO2(Glu), HC(NHCO2(Lys), HC(NHCO2(Lys)),
3.65−3.01 (m, 14H, CH2 N(CB-TE2A), H2CPhe, H2CNH(Lys),
H2CNH(Lys)), 2.74−2.41 (m, 20H, NCH2CH2(CB-TE2A), 2.41−
2.08 (m, 6H, H2CCO2(Glu), H2CCO2(linker), H2CCO2(linker)),
1.91−1.50 (m, 10H, CH2(CB-TE2A), H2CCH(Glu), H2CCH(Lys-
linker), H2CCH(Lys)), 1.56−1.22 (m, 16H, (CH2)2(Lys),
(CH2)2(Lys-Linker), (CH2)4(linker). ESMS m/z: 1222 [M + H]+.
HRESI+ MS: calcd for C60H91N11O16, 1221.6645; found, 1221.4951.
Copper Complexes of Ligand 3, 4, 5, 6, and 7. Copper
compounds [63/65 Cu]3−5 and[63/65 Cu]7 were prepared by the same
general procedure as presented for compound 3 below. [63/65Cu]6A−
B were prepared by same conditions as described for their
radiosynthesis.
[
63/65Cu]3. To a solution of Cu(NO3)2 (1 mg, 20 μmol in 100 μL)
in deionized water was added 3 (1 mg, 0.95 μmol) in 500 μL of 0.2 M
NaOAc. The resulting solution was heated in boiling water for 0.5 h.
The solution was purified by HPLC method 2. The retention time for
the product was 34 min. Yield: ∼50%. ESIMS m/z: 1115 [M + H]+.
HRMS: calcd for C46H70CuN9O17S, 1115.3906 [M + H]+; found,
1115.3828.
[
63/65Cu]4. The compound was purified by HPLC method 3.
Retention time for the product was at 22 min. Yield: ∼50%. ESIMS m/
z: 1192 [M + H]+. HRMS: calcd for C51H73CuN10O17S, 1192.4172 [M
+ H]+; found, 1192.4364.
[
63/65Cu]5. The compound was purified by HPLC method 4.
9,16,24-Trioxo-1-thioxo-1-((4-((4,7,10-tris(carboxymethyl)-
1-oxa-4,7,10-triazacyclododecan-2-yl)methyl)phenyl)amino)-
2,8,17,23,25-pentaazaoctacosane-7,22,26,28-tetracarboxylic
Acid, Compound 5. The compound was purified by HPLC (method
Retention time for the product was at 14 min. Yield: ∼60%. ESIMS m/
z: 1160 [M + H]+. HRMS: calcd for C48H76CuN9O18S, 1160.4247 [M
+ H]+; found, 1160.3828.
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[
63/65Cu]6A−B .The compound was purified by HPLC method 5.
under 20 μg/mL of puromycin to maintain PSMA expression. All cell
cultures were maintained in an atmosphere containing 5% carbon
dioxide (CO2) at 37.0 °C in a humidified incubator.
Retention time for the products were at 24 and 26 min. Yield: ∼50%.
ESIMS m/z: 1285 [M + H]+. HRMS: calcd for C60H91CuN11O16,
1284.5930 [M + H]+; found, 1284.5786.
Tumor Models. Animal studies were carried out in full compliance
with the regulations of the Johns Hopkins Animal Care and Use
Committee. Six-to-eight-week-old male, nonobese diabetic (NOD)/
severe-combined immunodeficient (SCID) mice (Johns Hopkins
Immune Compromised Core) were implanted subcutaneously (sc)
with PC3 PIP (PSMA+) and PC3 flu (PSMA−) cells (1 × 106 in 100
μL of HBSS (Corning Cellgro, Manassas, VA) at the forward right and
left flanks, respectively. Mice were imaged or used in ex vivo
biodistribution assays when the xenografts reached 5− 7 mm in
diameter.
Small-Animal PET Imaging and Analysis. Dynamic and whole-
body PET and CT images were acquired on an eXplore VISTA small-
animal PET (GE Healthcare) and an X-SPECT small SPECT/CT
system (Gamma Medica Ideas), respectively. For imaging studies, mice
were anesthetized with 3% and maintained under 1.5% isoflurane (v/
v). PET-CT Imaging studies were performed on NOD/SCID mice
bearing PSMA+ PC3 PIP and PSMA− PC3 flu tumors. Immediately
after intravenous injection of [64Cu]3−7, changes in radiotracer
accumulation were recorded over the whole body using an imaging
sequence consisting of eight frames for a total of 30 min with variable
dwell times as described previously.43,64 After dynamic imaging, whole-
body PET images (two bed positions, 15 min emission per bed
position) were acquired at the indicated time points after injection of
radiotracer. For binding specificity studies, a mouse was subcuta-
neously administered with a blocking dose of the known PSMA
inhibitor ZJ43 (50 mg/kg) at 30 min before the injection of [64Cu]6B,
and another mouse was injected with [64Cu]6B alone. After each PET
scan, a CT scan was acquired as 512 projections using a 50 keV beam
for anatomic coregistration. PET emission data were corrected for
decay and dead time and were reconstructed using the three-
dimensional ordered-subsets expectation maximization algorithm. Data
were displayed and analyzed using AMIDE software (http://
sourceforge.net/amide), and volume-rendered images were generated
using Amira 5.2.0 software (Visage Imaging Inc.; http://www.vsg3d.
com/amira).
[
63/65Cu]7. The compound was purified by HPLC method 6. Rt for
the product was at 13 min. Yield: ∼55%. ESIMS m/z: 1349 [M + H]+.
HRMS: calcd for C60H90CuN11O20, 1348.5650 [M + H]+; found,
1348.5671
64Cu Radiolabeling. Radioligands [64Cu]3−5 and 7 were
prepared by the same general method as described for [64Cu]3. For
each radiolabeling reaction, approximately 30−40 μg of ligand and in
200 mM NaOAc (purged under N2 for 2−3 min) was incubated with
3−4 mCi 64CuCl2 at pH 5.5−6 for 0.3 h in a water bath at 65 °C.
Radiolabeling was monitored by injecting aliquots of 20−40 μL of the
solution onto the HPLC. When the reaction reached completion, the
reaction mixture was diluted with 1 mL of water then loaded onto a
preparative HPLC for purification using method 2, Rt 41 min, for the
desired product and Rt 38 min for the free ligand. The radiolabeled
product [64Cu]3 was obtained in ∼60−70% radiochemical yield, and
the radiochemical purity was >98% as measured by ITLC (Gelman
ITLC strips, 10 mM EDTA). HPLC method 2 was used to purify the
radiolabeled product [64Cu]3. Rt 34 min for the desired product and Rt
42 min for the free ligand. The specific activity of the probe was 2.9−
9.1 GBq/μmol (n = 4). The acidic eluate was neutralized with 50 μL of
1 M Na2(CO3), and the volume of the eluate was reduced under
vacuum to dryness. The solid residue was diluted with saline to the
desired radioactivity concentration for biodistribution and imaging
studies. HPLC method 3 was used for [64Cu]4, Rt = 20−26 min for
[64Cu]4 and Rt = 15 min for the free ligand 4. [64Cu]4 was isolated in
two peaks, Rt 20−22 min for the first fraction and 23−26 min for the
second fraction. On the basis of the initial results (in PET imaging
both fractions displayed similar in vivo distributions), only the second
fraction was used for all studies. HPLC method 4 was used for [64Cu]
5. Retention times for [64Cu]5 and free ligand were and 14.5 and 9.5
min, respectively. The radiotracer [64Cu]7 was purified by HPLC
method 6. Rt for [64Cu]7 was at 13 min.
For [64Cu]6A−B, radiolabeling was done in a boiling water bath for
1 h by incubation of 64CuCl2 with the corresponding ligand using 0.2
NaOAc buffer at pH = 7.5−8. HPLC method 5, Rt = 24.9 min (A) and
26.8 min (B) for the desired products and Rt = 19.8 min (A) and 21.5
min (B) for the free ligands. The specific activity of the probe was 2.9
GBq−12.84 GBq/μmol.
Ex Vivo Biodistribution. Mice bearing PSMA+ PC3 PIP and
PSMA− PC3 flu xenografts were injected via the tail vein with 740
kBq (20 μCi) of 64Cu in 200 μL of saline. At 30, 60, 240, and 240 or
300 min postinjection, the mouse was sacrificed by cervical dislocation
and the blood immediately collected by cardiac puncture. The heart,
lungs, liver, stomach, pancreas, spleen, fat, kidney, muscle, small and
large intestines, urinary bladder, PSMA+ PC3 PIP and PSMA− PC3
flu tumors were collected. Each organ was weighed, and the tissue
radioactivity was measured with an automated gamma counter (1282
Compugamma CS, Pharmacia/LKBNuclear, Inc., Mt. Waverly,
Victoria, Australia). The % ID/g was calculated by comparison with
samples of a standard dilution of the initial dose. All measurements
were corrected for decay.
NAALADase Assay. The PSMA inhibitory activities of 3−7 and
the corresponding copper-labeled analogues were determined using a
fluorescence-based assay according to a previously reported procedure.
28 Briefly, lysates of LNCaP cell extracts (25 μL) were incubated with
the inhibitor (12.5 μL) in the presence of 4 μM N-acetylaspartylglu-
tamate (NAAG) (12.5 μL) for 120 min. The amount of the glutamate
released by NAAG hydrolysis was measured by incubating with a
working solution (50 μL) of the Amplex Red glutamic acid kit (Life
Technologies, Grand Island, NY) for 60 min. Fluorescence was
measured with a VICTOR3 V multilabel plate reader (Perkin-Elmer
Inc., Waltham, MA) with excitation at 490 nm and emission at 642
nm. Inhibition curves were determined using semilog plots, and IC50
values were determined at the concentration at which enzyme activity
was inhibited by 50%. Enzyme inhibitory constants (Ki values) were
generated using the Cheng−Prusoff conversion.69 Assays were
performed in triplicate. Data analysis was performed using GraphPad
Prism version 4.00 for Windows (GraphPad Software, San Diego,
California).
Cell Lines. Sublines of the androgen-independent PC3 human
prostate cancer cell line, originally derived from an advanced androgen
independent bone metastasis, were used. These sublines have been
modified to express high (PC3 PIP) or possess low (PC3 flu) levels of
PSMA and were generously provided by Dr. Warren Heston
(Cleveland Clinic). PSMA-expressing (PC3 PIP), nonexpressing
(PC3 flu) PCa cell lines, were grown in RPMI 1640 medium
(Corning Cellgro, Manassas, VA) containing 10% fetal bovine serum
(FBS) (Sigma-Aldrich, St.Louis, MO) and 1% penicillin−streptomycin
(Corning Cellgro, Manassas, VA) and PC-3 PIP cells were grown
Data Analysis. Data are expressed as mean standard deviation
(SD). Prism software (GraphPAD, San Diego, California) was used to
determine statistical significance. Statistical significance was calculated
using a paired t test. A P-value <0.0001 was considered significant.
ASSOCIATED CONTENT
* Supporting Information
■
S
Detailed spectral data and supporting PET-CT blocking study
image. This material is available free of charge via the Internet
at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
■
*For S.R.B.: phone, 410-955-8697; fax, 410-614-3147; E-mail,
sray9@jhmi.edu; address, Johns Hopkins Medical Institutions,
1550 Orleans Street, 4M07 CRB II, Baltimore, Maryland
21231, United States.
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Article
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*For M.G.P.: phone, 410-955-2789; fax, 443-817-0990; E-mail,
mpomper@jhmi.edu; address, Johns Hopkins Medical Institu-
tions, 1550 Orleans Street, 492 CRB II, Baltimore, Maryland
21231, United States.
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Wong, V. Correlation of PSMA ADC exposure with reduction in
tumor growth rate determined using serial PSA measurements from a
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A.; Low, S.; McDonnell, K.; Peeke, E.; Retnarajan, B.; Sabnis, A.;
Schnipper, E.; Song, J. J.; Song, Y. H.; Summa, J.; Tompsett, D.;
Troiano, G.; Van Geen Hoven, T.; Wright, J.; LoRusso, P.; Kantoff, P.
W.; Bander, N. H.; Sweeney, C.; Farokhzad, O. C.; Langer, R.; Zale, S.
Preclinical development and clinical translation of a PSMA-targeted
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the University of Wisconsin Cyclotron Research
Group for providing [64Cu]CuCl2. We also thank Nordion and
Macrocyclics, Inc., for providing PCTA-Bn-SCN and Oxo-
DO3A-Bn-SCN chelating agent, Drs. Cara Ferreira and Russell
Redshaw for helpful discussion, and James Fox, Gilbert Green,
and Dr. Yuchuan Wang for assistance with imaging and image
analysis. We are grateful for the following sources of support:
K25 CA148901, NIH NCI U54CA151838, NCI CA134675,
and NCI R01 CA093375.
ABBREVIATIONS USED
■
PSMA, prostate-specific membrane antigen; GCPII, glutamate
carboxypeptidase II; NAALADase, N-acetylated-α-linked acidic
dipeptidase; PET, positron emission tomography; SPECT,
single photon emission computed tomography; NOTA, 1,4,7-
triazacyclononane-1,4,7-triacetic acid; CB-TE2A, 4,11-bis-
(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane;
PCTA, 3,6,9,15-tetraazabicyclo[9.3.1]-pentadeca-1(15),11,13-
triene)-3,6,9-triacetic acid ; oxo-DO3A, oxa-4,7,1-tetraazacyclo-
dodecane-4,7,10-triacetic acid; DOTA, 1,4,7,10-tetraazacyclo-
dodecane-N,N′,N″,N‴-tetraacetic acid
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