Effect of chelators on the pharmacokinetics of 99mTc-labeled imaging agents for the prostate-specific membrane antigen (PSMA)

Article abstract of DOI:10.1021/jm400823w

Technetium-99m, the most commonly used radionuclide in nuclear medicine, can be attached to biologically important molecules through a variety of chelating agents, the choice of which depends upon the imaging application. The prostate-specific membrane antigen (PSMA) is increasingly recognized as an important target for imaging and therapy of prostate cancer (PCa). Three different ^99mTc-labeling methods were employed to investigate the effect of the chelator on the biodistribution and PCa tumor uptake profiles of 12 new urea-based PSMA-targeted radiotracers. This series includes hydrophilic ligands for radiolabeling with the [^99mTc(CO)₃] ⁺ core (L8-L10), traditional N_xS_y-based chelating agents with varying charge and polarity for the ^99mTc-oxo core (L11-L18), and a ^99mTc-organohydrazine-labeled radioligand (L19). ^99mTc(I)-Tricarbonyl-labeled [^99mTc]L8 produced the highest PSMA+ PC3 PIP to PSMA- PC3 flu tumor ratios and demonstrated the lowest retention in normal tissues including kidney after 2 h. These results suggest that choice of chelator is an important pharmacokinetic consideration in the development of ^99mTc-labeled radiopharmaceuticals targeting PSMA.

Full text of DOI:10.1021/jm400823w

Article
pubs.acs.org/jmc
Effect of Chelators on the Pharmacokinetics of 99mTc-Labeled
Imaging Agents for the Prostate-Specific Membrane Antigen (PSMA)
Sangeeta Ray Banerjee,*,† Mrudula Pullambhatla,† Catherine A. Foss,† Alexander Falk,† Youngjoo Byun,‡
Sridhar Nimmagadda,† Ronnie C. Mease,† and Martin G. Pomper*,†
†Russell H. Morgan Department of Radiology and Radiological Science, Johns Hopkins University, School of Medicine, Baltimore,
Maryland 21287, United States
‡College of Pharmacy, Korea University, 2511 Sejong-ro, Jochiwon-eup, Yeongi-gun, Chungnam 339-700, South Korea
S
* Supporting Information
ABSTRACT: Technetium-99m, the most commonly used radionuclide in nuclear medicine,
can be attached to biologically important molecules through a variety of chelating agents, the
choice of which depends upon the imaging application. The prostate-specific membrane
antigen (PSMA) is increasingly recognized as an important target for imaging and therapy of
prostate cancer (PCa). Three different 99mTc-labeling methods were employed to investigate
the effect of the chelator on the biodistribution and PCa tumor uptake profiles of 12 new
urea-based PSMA-targeted radiotracers. This series includes hydrophilic ligands for
radiolabeling with the [99mTc(CO)3]+ core (L8−L10), traditional NxSy-based chelating
agents with varying charge and polarity for the 99mTc-oxo core (L11−L18), and a 99mTc-
organohydrazine-labeled radioligand (L19). 99mTc(I)-Tricarbonyl-labeled [99mTc]L8 pro-
duced the highest PSMA+ PC3 PIP to PSMA− PC3 flu tumor ratios and demonstrated the
lowest retention in normal tissues including kidney after 2 h. These results suggest that choice
of chelator is an important pharmacokinetic consideration in the development of 99mTc-
labeled radiopharmaceuticals targeting PSMA.
of radiohalogenated, urea-based, low-molecular-weight inhib-
itors of PSMA to image PSMA expression in prostate tumor
xenografts.8,9 The SPECT agents [123I]MIP-1072 and
INTRODUCTION
■
Technetium-99m (half-life, 6 h; γ-energy, 140.5 keV) is widely
used in nuclear medicine because of its nearly ideal photon
energy for single photon emission computed tomography
(SPECT), low dose burden to the patient, low cost, ready
availability, and synthetic tractability for incorporating into
biological targeting agents. Despite the wide availability of PET
isotopes (such as 18F, 62/64Cu, and 68Ga) in Western countries,
99mTc remains the radionuclide of choice for development of
diagnostic radiotracers in most developing countries and is the
most heavily utilized diagnostic medical isotope.1 Adding
further to its convenience, 99mTc-labeled biotargeting agents
can be prepared via a commercially available kit. A variety of
well-established chelating agents is available for incorporating
99mTc into biotargeting agents, however, the effects of various
chelators and the composition of the 99mTc core (inherent
functionalities attached to 99mTc) on the pharmacokinetics of
the parent compounds have not been well characterized for
specific indications.2
Prostate cancer (PCa) is the most commonly diagnosed
malignancy with few options for molecular imaging due to its
relatively low metabolism. Prostate-specific membrane antigen
(PSMA), an integral membrane protein, is increasingly
recognized as a viable target for imaging and therapy of
PCa.3,4 Elevated expression of PSMA is associated with
metastasis,5 androgen independence,6 and progression7 of
PCa. We and others have previously demonstrated the ability
[
123I]MIP-1095 and the PET agent [18F]DCFBC have
demonstrated promise by detecting both bone and lymph
node metastases in clinical studies.10,11 Recently, we and others
have extended that work to include the radiometal 99mTc via
coordinated, 99mTc(I) tricarbonyl,12−16 or 99mTc(V)-oxo17,18
moieties. Generally, to retain binding affinity to PSMA, a linker
was required between the PSMA-targeting moiety and the
metal chelator.16 While changes in the linker are known to
affect the biodistribution of these agents,16,17 the effect of
various chelators and related 99mTc-labeled cores on the
pharmacokinetics of compounds of this class have not been
well characterized.
Here we expand upon our earlier work with urea-based,
PSMA-targeted imaging agents to address the effect of various
common chelators of 99mTc on the pharmacokinetics and
tumor uptake in a small series of new imaging agents. Here we
used three different 99mTc core complexes and related chelating
agents for comparison: (1) [99mTc(CO)3]+ core using a lysine-
based tridentate chelator, demonstrated in [99mTc]L8−L10, (2)
the 99mTc-oxo ([99mTcO]3+) core as in [99mTc]L11−L18,
which is most frequently used for radiolabeling of biomolecules
with 99mTc, and (3) 99mTc-organohydrazine [99mTcNHNR]2+ as
Received: April 6, 2013
Published: June 25, 2013
© 2013 American Chemical Society
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Figure 1. 99mTc-Labeled inhibitors of PSMA.
in [99mTc]L19, which is of particular interest due to its high
99mTc labeling efficiency (Figure 1). By altering the chelators
for 99mTc, the compounds produced consequently demon-
strated differences in overall charge, lipophilicity, stability, and
affinity, which we reasoned would alter their pharmacokinetics.
Additionally, we briefly investigated the effect of aromatic
substituents in the linker moiety on pharmacokinetics.
as shown in Scheme 1. In particular, a click reaction was
performed between commercially available fluorenylmethox-
a
Scheme 1. Synthesis of L8 and ReL8
Imaging and biodistribution studies in NOD/SCID mice
harboring PCa xenografts demonstrate that both tricarbonyl
and 99mTc-oxo complexes have favorable pharmacokinetics over
the HYNIC-conjugated compound. Between the tricarbonyl
and 99mTc-oxo complexes, tricarbonyl complex [99mTc]L8
exhibited superior tumor uptake and tumor-to-muscle and
tumor-to-blood ratios that are suitable for clinical translation.
Our studies also show that choice of chelating agent
significantly affects the stability, affinity, lipophilicity, and
ultimately pharmacokinetics of the final 99mTc-labeled radio-
ligands, suggesting that these results may have implications in
the synthesis of 99mTc-labeled imaging agents in general.
RESULTS
■
Chemical and Radiochemical Synthesis. Chemical and
radiochemical syntheses are shown in Schemes 1−5. Com-
pound L8 contains 1,4-disubstituted 1,2,3-triazole function-
alized lysine as a chelating agent. 99mTc labeling of this class of
triazole containing chelators has recently been developed by
Schibli’s group19 and was prepared through click chemistry20,21
a
(i) N-Boc-propargylglycine, Cu(OAc)2, sodium ascorbate, H2O/t-
BuOH (1:1), rt; (ii) 20% piperidine/DMF; (iii) 2, DMF, DIEA, rt;
(iv) TFA/CH2Cl2/TES; (v) [Re(CO)3(H2O)3]Br, MeOH/H2O.
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a
ycarbonyl (Fmoc)-Lys(azide)-OH and Boc-Gly-propargyl-OH
in the presence of a catalytic amount of Cu(OAc)2 and sodium
ascorbate and Tris-[(1-benzyl-1H-1,2,3-triazol-4-yl) methyl]-
amine (TBTA) at ambient temperature followed by Fmoc
removal to provide 1 in 45% yield. L8 was obtained upon
coupling of 1 with the NHS ester of urea-suberate 2,22 followed
by removal of the Boc group using TFA/CH2Cl2/triethylsilane
(TES) (1/1/0.05) in ∼60% yield. The rhenium tricarbonyl
complex (ReL8) was synthesized by using [Re(CO)3(H2O)3]-
Br23 as shown in Scheme 1. The monopyridyl monoacid lysine
chelating agent was prepared in a multistep synthesis as
outlined in Scheme 2. The acid group of Fmoc-Lys(Boc)-
Scheme 3. Synthesis of L10
a
Scheme 2. Synthesis of L9
a
(i) Imidazole-2-aldehyde, NaBH(OAc)3, CH3CO2H, NaBH(OAc)3,
dichloroethane; (ii) 20% piperidine/DMF; (iii) 2, DIEA, DMF.
free ε-amine of lysine was then conjugated with 2, followed by
Fmoc removal and conjugation of the α-amine with the NHS
ester of mercaptoacetic acid24 to produce L11 as shown in
Scheme 4.
a
Scheme 4. Synthesis of L11
a
(i) TsOH, EtOH EtOAc, rt; (ii) py-2-aldehyde, NaBH(OAc)3, DCE,
a
rt; (iii) t-butyl bromoacetate KI, DIEA, DMF; (iv) 20% piperidine/
DMF; (v) TFA, CH2Cl2, TIS; (vi) 2 DMF, DIEA.
(i) 20% piperidine in DMF; (ii) Fmoc-Gly-OH, HBTU, HOBT,
DIEA, DMF; (iii) 20% piperidine in DMF; (iv) Fmoc-Gly-OH,
HBTU, HOBT, DIEA, DMF; (v) TFA/TIS/CH2Cl2; (vi) 2, DIEA,
DMSOk, rt; (vii) S-trityl-CH2CO2-NHS, DIEA, DMF.
CO2H was protected using p-methoxy-benzyl chloride and
Cs2CO3 to obtain 3 in 70% yield.16 Selective removal of the
Boc group using p-toluenesulfonic acid in the presence of
ethanol at ambient temperature provided 4 in 60% yield.
Reductive amination of 4 with pyridine-2-aldehyde provided 5
in 40% yield. Alkylation of 5 by bromo-t-butyl-acetate at 25 °C
produced 6 in moderate yield (33%). Removal of the Fmoc
group by 20% piperidine/DMF followed by removal of the
PMB groups and the tert-butyl group by TFA/CH2Cl2 at 25 °C
afforded the desired chelating agent 7, which was reacted with 2
to produce L9 in a yield of 60%. Compound L10 was prepared
in two steps as described in Scheme 3. Reductive amination of
imidozole-2-aldehyde with Fmoc-Lys-OH in the presence of
NaBH(OAc)3 and acetic acid followed by Fmoc removal
provided chelating agent 8 in ∼30% yield. Chelating agent 8
was reacted with 2 to provide the PSMA-targeting ligand L10
in ∼50% yield.
Compound L19 was generated through a well-established
labeling approach using the HYNIC chelating agent (Scheme
5). Commercially available HYNIC(Boc)-NHS was conjugated
to compound 10,25 to produce L19 in 90% yield.
Radiotracers [99mTc]L8−L10 were synthesized as 99mTc-
(CO3) complexes using the same general method as described
a
Scheme 5. Synthesis of L19
A generalized Fmoc-based solid phase peptide synthesis
strategy was developed with a combination of solution phase
chemistry as required to prepare ligands L11−L18. Commer-
cially available Fmoc-Lys(Boc)-Wang resin was conjugated with
required amino acid residues, after which the compound was
cleaved from the resin by an ice-cold mixture of TFA/CH2Cl2/
triethyl silane (TES) (1/1/0.01) for 1 h at ambient
temperature. After isolating the resin cleaved product 9, the
a
(i) 10, DIEA, DMF; (ii) TFA/CH2Cl2/TIS.
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previously by us for the preparation of [99mTc]L1 using the
tricarbonyl labeling Isolink kit.16 These new radioligands were
obtained in reasonable radiochemical yields (70−95%) and in
high radiochemical purities (>98%) as determined by HPLC,
with a specific activity >411 GBq/μmol (1.1 × 104 mCi/ μmol)
upon reaction with [99mTc(CO)3]+ at ligand concentrations of
10−6 M at 95 °C (boiling water bath) for 30 min.
mice (Figures 2−4, Supporting Information Figures S2−S7)
bearing PSMA+ PC3 PIP and PSMA− PC3 flu xenografts in
opposite flanks at 2 h postinjection. Pharmacokinetics derived
from the images were used to determine which compound(s)
would be further evaluated in tissue biodistribution studies.
Irrespective of charge and lipophilicity, radioligands [99mTc]
L8−L18 enabled visualization of PSMA+ PC3 PIP tumor and
the kidneys. Renal uptake of the radiotracers is partially due to
their route of excretion as well as to specific uptake from the
expression of PSMA in mouse proximal tubules.29 In vivo
images of the neutral complex, [99mTc]L8, were promising
because of comparatively low background within the gastro-
intestinal tract and gallbladder compared to the previously
published, positively charged compound, [99mTc]L1. SPECT-
CT images acquired at 30 min, 2 and 6 h postinjection of
Radiolabeling of L11−L18 was performed following a
general literature procedure.26 Ligands were dissolved in 0.5
M ammonium acetate with 5−10% DMSO and reacted with
disodium tartrate and a solution of ascorbic acid and SnCl2,
−
followed by addition of 99mTcO4 and left at ∼90−95 °C for
10−15 min. Radiolabeled products were obtained in >80%
yield for all compounds, in >99% radiochemical purity with
specific activity >411 GBq/μmol (1.1 × 104 mCi/μmol). Two
radiolabeled peaks were isolated for all three MAG-2
conjugated radioligands [99mTc]L11, [99mTc]L12, and [99mTc]
L13, whereas for all MAG-3 and MAS-3 conjugated radio-
tracers, only a single peak was isolated during radiolabeling.
Interestingly, for all ligands except for L16, radiolabeling went
smoothly using the S-trityl protected version of the ligand.
Although both the protected and deprotected versions
ultimately provide the same radiolabeled product, we used
the protected starting materials to enable easier UV detection
and separation by HPLC. A single set of HPLC conditions
could be used to isolate radioligands [99mTc]L11−L18.
Moreover, the S-trityl protected ligands proved stable in
solution until subjected to radiolabeling conditions. Radio-
labeling of the HYNIC adduct was performed according to a
literature procedure by treating with tricine and SnCl2 at
ambient temperature for 30 min.27
[
99mTc]L8 (Supporting Information Figure S2), showed clear
PSMA+ PC3 PIP tumor uptake as early as 30 min
postinjection. At 30 min and 2 h postinjection, the most
visible tissues were PSMA+ PC3 PIP tumor and kidneys, with
some accumulation of radioactivity observed in the liver,
gallbladder, gastrointestinal tract, and urinary bladder. The
radioactivity in the gallbladder and kidneys cleared significantly
by 6 h, however, there was mild, residual retention of
radioactivity within the gastrointestinal tract. Although
[
99mTc]L9 and [99mTc]L10 demonstrated high PSMA+ PC3
PIP tumor uptake, they also showed significant accumulation
within gallbladder and the gastrointestinal tract at 2 h
postinjection (Figure 2). Accordingly, [99mTc]L9 and [99mTc]
L10 were considered to have provided inferior images
compared to [99mTc]L8 and our previous lead compound,
[
99mTc]L1.16
In Vitro Binding and Cell Uptake. All compounds
demonstrated high binding affinity to PSMA with Ki values
ranging from 0.03 to 16.30 nM (Table 1). We also synthesized
a
Table 1. PSMA Inhibitory Activity
compd
Ki
95% CI (Ki) (nM)
L8
1.12
0.35
12.60
16.30
0.03
0.13
1.91
7.99
0.18
0.10−11.62
0.25−0.54
10.46−15.20
ReL8
L9
a
a
a
a
L10
L11
L12
L13
L14
L15
L16
L17
L18
L19
ZJ43
8.78−30.32
0.02−0.06
0.08−0.20
1.54−2.37
2.05−31.14
0.10−0.32
0.02−0.14
0.11−0.30
2.24−3.33
0.96−3.17
0.31−0.62
Figure 2. Whole body SPECT-CT imaging of PSMA+ PC3 PIP and
PSMA− PC3 flu tumor-bearing mice with tricarbonyl compounds
0.048
0.17
2.73
1.74
0.43
[
99mTc]L8, [99mTc]L9, and [99mTc]L10 at 2 h postinjection,
respectively. Mice were injected with ∼37 MBq (1 mCi) of
radiopharmaceutical IV. PIP = PC3 PSMA+ PC3 PIP (solid arrow),
flu = PSMA− PC3 flu (unfilled arrow), GB = gallbladder, GI =
gastrointestinal tract, K = kidney, B = bladder, L = left, R = right. All
images are decay-corrected and adjusted to the same maximum scale.
a
Separate experiment, ZJ43, (Ki 11.81 nM, 95% CI(10.31−13.53)).
an analogue of L8 that incorporates Re and observed a 3-fold
improvement in binding affinity for ReL8 over L8 (Table 1).
Comparative cell uptake involving [99mTc]L8 revealed ∼100
fold higher uptake in PSMA+ PC3 PIP cells relative to PSMA−
PC3 flu cells. This uptake in PIP cells could be blocked by
treatment with 10 μmol of the known, high-affinity PSMA
inhibitor, ZJ43 (Supporting Information Figure S1).28
SPECT-CT images of the negatively charged 99mTc-oxo-
labeled radioligands [99mTc]L11−L15 (Figure 3) and [99mTc]
L16 (Figure 4), and neutral radioligands [99mTc]L17 and
[
99mTc]L18 (Figure 4) demonstrated high radiotracer accumu-
lation within PSMA+ PC3 PIP tumor and kidneys at 2 h
postinjection. Furthermore, among [99mTc]L11, [99mTc]L15,
and [99mTc]L18 (Figure 3, Supporting Information Figures S3,
S4 and S6), retention of radioactivity within PSMA target sites
Small Animal SPECT-CT Imaging. Whole body SPECT-
CT images were obtained for [99mTc]L8−L19 in male SCID
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Figure 3. Whole-body SPECT-CT imaging of PSMA+ PC3 PIP and PSMA− PC3 flu tumor-bearing mice with [99mTc]L11A, [99mTc]L11B, [99mTc]
L12A, [99mTc]L13A, [99mTc]L14, and [99mTc]L15 at 2 h postinjection, respectively. Mice were injected with ∼37 MBq (1 mCi) of
radiopharmaceutical IV. PIP = PSMA+ PC3 PIP (filled arrow), flu = PSMA− PC3 flu (unfilled arrow), GB = gallbladder, GI = gastrointestinal tract,
K = kidney, B = bladder, L = left, R = right. All images are decay-corrected and adjusted to the same maximum scale.
was observed even at 18 h, suggesting the opportunity for
protracted imaging using these agents.
As a further test of binding specificity, we imaged animals
administered [99mTc]L11 after pretreating them with 50 mg/kg
of ZJ43. ZJ43 proved capable of blocking the binding of
[
99mTc]L11 (Supporting Information Figure S5), not only
within the tumor but also within the renal cortex and salivary
glands, confirming that uptake observed in these tissues is
PSMA-mediated.
Among the MAG-2-chelated ligands, L11−L13, which
possess different lipophilicities due to the presence of zero,
one, and two Phe residues on the linker, respectively,
considerable differences in nontarget tissue uptake was
observed for their radiolabeled counterparts (Figure 3). High
gallbladder, liver, and gastrointestinal uptake were observed for
Figure 4. Whole body SPECT-CT imaging of PSMA+ PC3 PIP and
PSMA− PC3 flu tumor-bearing mice with [99mTc]L16, [99mTc]L17,
and [99mTc]L18 at 2 h postinjection, respectively. Mice were injected
with ∼37 MBq (1 mCi) of radiopharmaceutical IV. PIP = PSMA+
PC3 PIP (filled 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 scale.
[
99mTc]L13 compared to [99mTc]L11. Also, as mentioned
above, [99mTc]L11−L13 were obtained as two different
diastereomers. Both radiolabeled isomers were investigated in
SPECT imaging studies for [99mTc]L11 (Figure 3 and
Supporting Information Figure S3). No significant difference
was observed with respect to the tumor uptake, nontarget tissue
uptake, or clearance between the isomeric compounds.
a
Table 2. Biodistribution of [99mTc]L8 in Tumor Bearing Mice
0.5 h
1 h
2 h
5 h
blood
1.35 1.22
0.65 0.26
2.47 0.83
28.85 17.93
0.73 0.16
1.08 0.18
21.11 8.49
1.08 0.49
122.70 14.73
0.71 0.27
2.26 1.27
0.57 0.15
1.72 0.86
28.31 4.38
0.50 0.07
53.95 1.61
32.14 4.75
42.87 29.34
0.46 0.20
0.52 0.23
1.78 0.29
11.36 1.57
0.41 0.21
0.43 0.12
12.28 2.99
0.66 0.06
119.15 0.01
0.70 0.52
1.35 0.19
0.29 0.06
7.70 0.30
28.05 2.04
0.24 0.06
112.59 30.47
41.24 26.57
38.44 17.81
0.19 0.05
0.22 0.14
0.92 0.14
6.11 0.66
0.17 0.05
0.25 0.12
4.42 1.68
0.33 0.12
116.2 14.2
0.23 0.16
0.69 0.20
0.81 0.51
1.22 0.33
26.29 7.45
0.19 0.08
103.90 32.36
108.72 28.78
103.99 57.00
0.13 0.05
0.08 0.01
0.30 0.09
3.24 0.39
0.20 0.13
0.10 0.06
1.83 1.52
0.12 0.04
55.31 1.15
0.08 0.04
0.14 0.05
1.31 0.9
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
1.11 0.56
23.22 6.02
0.07 0.01
307.24 25.25
178.00 79.79
141.58 88.36
a
Values expressed are in % ID/g standard deviation. N = 4 for all tissues.
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a
Table 3. Biodistribution of [99mTc]L11 in Tumor-Bearing Mice
30 min
1 h
2 h
5 h
blood
1.09 0.04
2.34 0.29
7.05 0.95
1.73 0.13
1.68 0.32
3.83 0.45
88.02 22.76
3.45 0.54
139.49 12.80
8.55 0.84
1.79 0.30
1.53 0.40
1.54 0.25
5.21 1.98
35.30 7.04
2.39 0.80
15.62 4.44
32.55 6.97
20.23 5.04
0.47 0.03
1.90 1.28
7.53 0.40
1.25 0.24
1.62 0.27
2.97 1.05
97.78 29.74
2.65 1.04
160.90 31.40
8.69 4.24
2.32 1.45
2.33 2.49
1.04 0.33
3.14 2.34
42.46 4.37
1.41 0.61
42.85 0.29.35
54.92 30.48
48.60 48.60
0.30 0.03
1.31 0.68
4.54 3.31
0.81 0.08
0.93 0.18
2.82 0.64
77.57 26.42
0.65 1.31
159.18 19.50
21.52 17.08
1.18 0.50
0.80 0.36
0.51 0.34
2.22 0.63
33.59 3.83
0.77 0.22
46.58 15.90
111.74 10.84
33.93 17.74
0.45 0.34
0.61 0.28
3.39 1.94
0.64 0.37
0.66 0.06
1.61 0.21
45.45 25.43
1.09 1.26
162.26 11.25
33.68 13.01
0.38 0.33
0.44 0.18
0.73 0.43
1.35 0.56
30.00 9.60
0.80 0.43
46.04 27.92
43.13 37.85
57.91 48.65
heart
lung
liver
stomach
pancreas
spleen
fat
kidney
lymph node
muscle
small intestine
large intestine
bladder
PC3 PIP
PC3 flu
PIP: flu
PIP: blood
PIP: muscle
a
Values expressed are in % ID/g standard deviation. N = 4 for all tissues.
a
Table 4. Biodistribution of [99mTc]L15 in Tumor-Bearing Mice
30 min
1 h
2 h
5 h
blood
0.53 0.15
0.72 0.23
2.83 0.81
0.62 0.23
1.01 0.83
1.23 0.56
24.05 9.01
0.85 0.60
242.68 36.55
0.83 0.54
0.46 0.07
0.48 0.04
1.09 0.43
20.52 2.12
0.41 0.06
48.18 7.67
42.49 21.24
63.69 80.92
0.54 0.07
0.72 0.09
2.59 0.80
0.39 0.05
1.56 0.65
2.36 2.43
25.66 4.99
1.45 0.44
346.60 28.12
0.86 0.02
0.53 0.36
0.47 0.21
1.24 0.86
23.44 3.82
0.43 0.09
57.40 7.12
42.57 11.64
18.65 16.57
0.24 0.35
0.17 0.11
1.58 0.45
0.26 0.09
1.40 0.92
0.54 0.20
7.55 2.14
0.10 0.19
270.08 44.35
0.13 0.31
0.20 0.18
0.06 0.12
0.25 0.47
18.27 3.35
0.29 0.13
70.14 24.93
67.0328 0.00
73.65 0.00
0.19 0.25
0.23 0.06
1.80 0.14
0.24 0.07
0.45 0.23
0.55 0.09
10.10 2.34
0.35 0.61
261.65 26.90
0.34 0.12
0.38 0.18
0.25 0.05
2.42 1.41
28.01 5.81
0.38 0.19
92.48 40.51
63.08 19.84
85.42 27.06
heart
lung
liver
stomach
pancreas
spleen
fat
kidney
muscle
sml. intetine
lrg intestine
bladder
PC3 PIP
PC3 flu
PIP: flu
PIP: blood
PIP: muscle
a
Values expressed are in % ID/g standard deviation. N = 4 for all tissues.
Compounds [99mTc]L14 and [99mTc]L15 exhibited high
radiotracer concentration both within PSMA+ tumor and
the % ID/g uptake values in selected organs for these
compounds at 30 min, 1 h, 2 h, and 5 h postinjection. All
compounds showed clear PSMA-dependent binding in PSMA+
PC3 PIP tumor xenografts, with [99mTc]L8, [99mTc]L11, and
kidneys, similar to the distribution profile observed with
[
99mTc]L11. Similarly, [99mTc]L17 and [99mTc]18 also
demonstrated similar distribution profiles to [99mTc]L11 at 2
h postinjection. However, faster clearance from kidney than
from PSMA+ tumor was noted with [99mTc]L18 at 18 h
postinjection (Supporting Information Figure S6). SPECT-CT
images of [99mTc]L19 demonstrated no specific uptake in
PSMA+ tumor or kidneys (Supporting Information Figure S7)
and provided diffuse and indiscriminant uptake throughout the
tissues.
[
99mTc]L15 demonstrating high tumor uptake as early as 30
min postinjection and retention of radioactivity at this level up
to 5 h postinjection. For [99mTc]L8, highest tumor uptake was
observed at 30 min (28.3 4.4% ID/g). PSMA+ PC3 PIP to
PSMA− PC3 flu tumor uptake ratios were 53.9 1.6 and 307.2
24.2 at 30 min and 5 h, respectively. The distribution within
normal organs and tissues was also favorable, with low blood
and nonspecific tissue uptake and rapid clearance. The highest
nonspecific uptake observed was in the liver, 28.9 17.9% ID/
Biodistribution. On the basis of the SPECT-CT imaging
results, [99mTc]L8, [99mTc]L11, and [99mTc]L15 were further
assessed in a biodistribution assay using the same PSMA+ PC3
PIP and PSMA− PC3 flu tumor models.22 Tables 2−4 show
g, and spleen, 21.1
8.5% ID/g at 30 min postinjection.
However, those values decreased to 3.2 0.4% and 1.8 1.5%,
respectively, by 5 h. Kidney uptake was expectedly high and
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Table 5. Biodistribution of [for [99mTc]L11, [99mTc]L13, [99mTc]L14, [99mTc]L15, and [99mTc]L18 at 2 h Post-Injection in
a
Tumor-Bearing Mice
[
99mTc]L11
[
99mTc]L13
[
99mTc]L14
[
99mTc]L15
[
99mTc]L18
blood
0.42 0.08
2.26 0.64
6.68 1.27
1.44 0.12
1.10 0.36
3.45 0.17
47.90 17.12
4.82 0.95
134.92 8.61
1.13 0.41
1.08 0.33
0.93 0.43
3.12 0.98
26.81 1.94
0.89 0.09
30.32 2.81
66.99 17.84
25.70 9.14
0.35 0.18
1.36 0.94
4.14 1.91
0.80 0.27
0.60 0.15
1.82 0.64
49.91 21.99
2.16 0.83
142.4 12.20
0.94 0.21
0.97 0.20
1.16 0.66
1.66 0.72
32.02 15.78
0.42 0.07
83.12 45.46
92.25 22.53
48.40 32.08
0.18 0.04
1.62 0.60
6.06 0.44
1.11 0.13
0.82 0.06
3.12 0.39
75.29 28.42
5.54 3.31
143.42 17.97
1.28 0.43
0.90 0.30
0.84 0.38
2.16 0.73
28.67 5.84
0.69 0.29
46.81 18.49
165.13 53.21
22.84 11.16
0.24 0.04
1.02 0.41
4.43 0.51
0.67 0.15
0.82 0.23
1.66 0.24
45.43 9.82
3.60 0.83
226.44 28.03
0.86 0.29
0.44 0.13
0.61 0.07
1.74 0.93
24.37 3.12
0.50 0.12
53.45 17.32
103.40 11.73
32.88 14.71
0.49 0.06
0.63 0.25
3.05 0.94
0.76 0.20
0.49 0.19
1.96 1.20
26.40 8.95
4.67 0.01
198.0 34.12
0.43 0.15
0.42 0.13
0.65 0.34
2.61 0.27
33.59 3.20
0.43 0.11
80.14 12.87
69.17 3.94
82.79 19.99
heart
lung
liver
stomach
pancreas
spleen
fat
kidney
muscle
sml intest
lrg intest
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.
peaked at 122.7 14.7% ID/g at 30 min and decreased to 55.3
1.2% ID/g by 5 h.
higher than for [99mTc]L11 at all time points, with a peak value
at 1 h postinjection of 347.0 28.0% ID/g.
Table 3 shows the organ-related % ID/g of uptake for
[
99mTc]L11 and [99mTc]L15 were also compared to [99mTc]
[
99mTc]L11. Similar to [99mTc]L8, [99mTc]L11 showed high
L13, [99mTc]L14, and [99mTc]L18 in a single time point (2 h)
biodistribution study (Table 5). Syntheses and biodistributions
were performed on the same day as close to one another as
possible to minimize the changes in specific activity. The
biodistribution and imaging data reveal several important points
regarding the in vivo properties of these compounds. First, the
MAG-2 conjugated radiotracers [99mTc]L11 and [99mTc]L13
showed high levels of uptake in PSMA+ PC3 PIP tumor and
kidney, however, with [99mTc]L11 having slightly higher
nonspecific uptake observed in PSMA− PC3 flu tumor and
other organs which resulted in overall lower tumor-to-organ
ratios than for [99mTc]L13. However, in the imaging studies, we
noted considerable gastrointestinal uptake of [99mTc]L13,
which could not be captured in the limited tissue
biodistribution study. Imaging results alone indicate that
PSMA-dependent tumor uptake with 42.5 4.4% ID/g at 1 h
postinjection. Tumor uptake remained high, decreasing to 30.0
9.6% ID/g at 5 h. The PSMA+ PC3 PIP to PSMA− PC3 flu
ratio was 46.6 15.9 at 2 h. Although this is a lower value than
that seen for [99mTc]L8, it is similar to the values observed with
one of our original compounds, [99mTc]L1.16 The PSMA+ PC3
PIP tumor-to-muscle ratio for [99mTc]L11 reached a maximum
value of 61.7 48.6 at 5 h, about one-half the value for [99mTc]
L8. Renal uptake for [99mTc]L11 was high at all times and
peaked at 5 h at 162.3 11.3% ID/g, much higher than that
seen for [99mTc]L8. Nontarget organs, such as blood, heart,
liver, stomach, and pancreas, showed less uptake (∼2% ID/g at
1 h), which decreased to below 1% ID/g by 5 h. Compound
[
99mTc]L11 exhibited high lung uptake (7.1 1.0% ID/g at 30
[
99mTc]L11 demonstrates superior image contrast compared
min) compared to [99mTc]L8, which decreased to 3.4 1.9%
by 5 h. Interestingly, high spleen uptake of 97.8 30.0% ID/g
at 1 h was also observed, which decreased to 45.5 25.4% ID/
g by 5 h.
to [99mTc]L13. Second, between the MAG-2, MAG-3, and
MAS-3 conjugated radiotracers, all exhibited similar tumor
uptake (∼20% ID/g), however, [99mTc]L15, which employs a
MAS-3 chelator, exhibited ∼2-fold higher accumulation of
radioactivity within kidneys than did [99mTc]L11 or [99mTc]
L14, perhaps due to the presence of three polar hydrophilic
functional groups on the chelator backbone. Third, between the
hydrophilic [99mTc]L15 (MAS-3) and [99mTc]L18, the latter,
with two carboxylic acid groups from Asp residues on the
chelator side chain, showed higher tumor uptake and lower
accumulation of radioactivity in most normal tissues, including
spleen, resulting in higher tumor-to-organ ratios than for
Table 4 shows the % ID/g of uptake for MAS-3 chelated
[
[
99mTc]L15. Compound [99mTc]L15, similar to [99mTc]L8 and
99mTc]L11, demonstrated PSMA-dependent tumor uptake,
which peaked at 5 h at 28.0 5.8% ID/g. These values are
similar to those observed with [99mTc]L8 but lower than for the
MAG-2 conjugated radioligand, [99mTc]L11. However, PSMA+
PC3 PIP to PSMA− PC3 flu ratios for [99mTc]L15 were higher
than those for [99mTc]L11, achieving a maximum value of 92.5
40.5 at 5 h. PSMA+ PC3 PIP tumor-to-muscle ratios were
also higher, achieving a maximum value of 85.4 27.4 at 5 h as
opposed to 48.6 48.6 at 1 h, which was observed for [99mTc]
L11. Nontarget organs, except for spleen, demonstrated little to
no uptake (≤1% ID/g throughout the study). Spleen uptake for
[
99mTc]L15.
To investigate the cause of the high spleen uptake for the
99mTc-oxo compounds, two blocking studies were performed
on selected organs using normal (nontumor) male CD-1 mice
as shown in Table 6, employing both 2-(phosphonomethyl)-
pentanedioic acid (2-PMPA)30 and ZJ43 for [99mTc]L15.
Greater than 90% of uptake observed could be blocked in
[
99mTc]L15 was also lower than for [99mTc]L11, peaking at
25.7 5.0% ID/g at 1 h, decreasing to 10.1 2.3% ID/g by 5 h
postinjection. Kidney uptake for [99mTc]L15 was significantly
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Table 6. Biodistribution of [99mTc]L15 Using ZJ43 and 2-
PMPA as Blocking Agents in Healthy Male CD-1 Mice at 2 h
Postinjection
different well-established labeling strategies. For example,
[
99mTc(CO)3]+-labeled complexes are robust due to their
a
kinetic inertness and do not, in general, dissociate in vivo.
However, because of their organometallic nature, these
compounds tend to be lipophilic. 99mTc-oxo compounds, on
the other hand, are stable in the circulation but show increased
renal retention. As a result, we observed a clear shift in the
pharmacokinetics for the tricarbonyl complexes [99mTc]L8−
L10 in terms of liver uptake and the extent of hepatobiliary
excretion when compared to the polar 99mTc-oxo compounds.
Elevated renal radioactivity generally observed with 99mTc-oxo
complexes was further compounded by the high level of PSMA
expression in murine kidney.38 Changing the chelator also
allows manipulation of the overall charge, while molecular
weight and lipophilicity of these compounds can be altered with
minor modifications in the linker. In agreement with previously
published results on chelate stability and in vivo performance of
different bioconjugates such as affibodies39 and peptides40−42
using same the 99mTc-labeling core, we observed significant
changes in the biodistribution pattern mostly in nonspecific
tissue rather than in PSMA-specific tumor uptake.
[
99mTc]L15
[
99mTc]L15 + ZJ43
[
99mTc]L15 + 2-PMPA
blood
spleen
kidney
muscle
testis
0.27 0.02
6.34 2.01
0.21 0.02
0.14 0.05
1.54 0.20
0.51 0.54
0.09 0.01
0.22 0.02
0.10 0.01
1.69 0.72
0.34 0.02
0.14 0.08
105.21 10.92
0.49 0.17
0.82 0.10
a
Values expressed are in % ID/g standard deviation. N = 4 for all
tissues.
spleen, testis, and kidney, suggesting that the uptake in these
tissues is PSMA-mediated.
DISCUSSION
■
Previously, we reported 99mTc-labeled PSMA inhibitors with an
optimized linker length and obtained a high-affinity radioligand,
[
99mTc]L1 (Figure 1), using the single amino acid chelating
agent (SAAC) to incorporate 99mTc.16,31 Utilizing the same
linker construct, we have now synthesized and evaluated 12
new 99mTc-labeled PSMA-targeting radioligands (Figure 1),
which vary primarily with respect to lipophilicity and charge,
using well-established 99mTc labeling techniques.32,33 Our
previously published compound, [99mTc]L1, carries a positive
charge. To complement [99mTc]L1 and the rest of our previous
series that relied upon SAAC technology, we synthesized three
neutral Tc(I)-tricarbonyl labeled radioligands, [99mTc]L8−L10,
and seven negatively charged 99mTc-oxo labeled complexes,
SPECT imaging revealed that [99mTc]L8, the most hydro-
philic radioligand in the tricarbonyl series, had high and specific
tumor uptake in the PSMA+ PC3 PIP tumor and long tumor
retention of radioactivity, with nearly 82% of the initial uptake
(30 min) retained even at 5 h postinjection. This is a significant
improvement over the previous lead compound [99mTc]L1,
where radiotracer was nearly completely cleared from tumor by
5 h postinjection.16 Compound [99mTc]L8 showed low uptake
in normal tissues such as liver and gallbladder at late time
points. Rapid clearance observed from blood suggested that
there was no transchelation to blood proteins. Similarly rapid
clearance was also observed from liver, spleen, and kidney.
Because the tumor models were derived from isogenic human
PC3 PCa cells and are relatively uniform in size and location,43
we surmise that the overall neutral charge, small size of the
chelating agent, and relative hydrophilicity of L8 contributed to
the high and specific binding to PSMA with rapid clearance
from normal tissues.
[
99mTc]L11−L16. Of these, [99mTc]L11−L15 utilize the N3S
chelator. Radiotracers [99mTc]L11−L13 contain a previously
reported, mercaptoacetyl-Gly-Gly (MAG-2) chelating agent
along with hydrophobic substituents, such as Phe groups placed
on the linker, to investigate pharmacokinetic effects of these
modifications.34,35 Consistent with that previously described by
Liu et al., [99mTc]L11−L13 and [99mTc]L16 were obtained as
two diastereomers (syn and anti) due to the relative orientation
of the functional group (carboxylate or Phe residue) on the
chelator to the TcO core. Also, chelating agent mercaptoa-
cetyl-Gly-Gly-Gly (MAG-3) was used to synthesize [99mTc]
L14.34 We envisioned that introduction of hydroxyl groups may
improve clearance from nontarget tissues; therefore, we
synthesized a more hydrophilic version of this compound,
The most striking feature of the 99mTc-oxo series is the high
and prolonged uptake within spleen, which was not observed
for radioligands employing the tricarbonyl chelator. We
hypothesize that elevated spleen uptake and retention could
be related to binding to a homologue of PSMA, namely
glutamate carboxypeptidase III (GCPIII), which has recently
been described.44,45 GCPIII is highly expressed in spleen, testis,
and kidney46 and has been shown to be a target of some low
molecular weight antagonists of PSMA.47 Because none of
these antagonists are specific for GCPIII, our blocking studies
with 2-PMPA, which has demonstrated affinity to GCPIII, and
ZJ43 are not conclusive regarding GCPIII-mediated binding.
Blocking studies performed for [99mTc]L15 in blood, spleen,
and kidney do indicate GCPII- and/or GCPIII-mediated tissue
uptake. However, because blocking was >90% for both
compounds, a significantly greater element of blocking for 2-
PMPA could not be demonstrated, which may have suggested a
GCPIII-specific element. In a recent preclinical study, high
spleen and kidney uptake by a 68Ga-labeled Lys-Glu urea-based
PSMA PET agent using N,N′-bis[2-hydroxy-5-(carboxyethyl)-
benzyl]ethylenediamine-N,N′-diacetic acid (HBED-CC) as the
chelating agent has been reported.48 However, the first clinical
study of this agent demonstrated only moderate uptake within
spleen, which did not interfere with detection of putatively
[
99mTc]L15, with the mercaptoacetyl-Ser-Ser-Ser (MAS-3)
chelating group, which has three hydroxyl groups. Compound
[
99mTc]L16 employed a diamide dithiol (N2S2) chelating agent,
reported to form stable 99mTc-oxo complexes.35,36 Because
compounds [99mTc]L11−L16 form negatively charged 99mTc
complexes at physiological pH, to investigate the effect of this
charge on pharmacokinetics, we synthesized compounds
[
99mTc]L17 and [99mTc]L18, which are reported to form
neutral 99mTc-oxo complexes.37 Compounds [99mTc]L17 and
99mTc]L18 use Cys-Asp-Gly and Cys-Asp-Asp chelators,
[
respectively. We also synthesized compound [99mTc]L19
using the HYNIC core and tricine as a coligand for 99mTc
labeling.32 All of the compounds were equally synthetically
accessible and easy to purify and administer. The 12
compounds studied had similar affinities, in the low nanomolar
range, similar to the majority of the PSMA-binding imaging
agents we have developed to date.16,22,25
The rationale for the use of these different chelators was to
exploit the different coordination chemistry of 99mTc using
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PSMA+ lesions.49 These findings suggest that a species-related
difference, perhaps due to differential expression of GCPIII,
exists and that elevated uptake in some nontarget sites such as
spleen in preclinical rodent studies may not apply to clinical
studies.
any purification in the next step where the Fmoc group was removed
using 20/80 piperidine/DMF solution at rt to produce 1. Compound
1 was purified using C18 liquid chromatography column and a mobile
phase consisting of 80/20 H2O/CH3CN and lyophilized. Yield: ∼45%.
To a solution of compound 2 (0.05 g, 0.09 mmol in 250 μL DMF)
was added 1 (0.35 g, 0.09 mmol in 50 μL) and was stirred at rt for 2 h.
The solution was removed under vacuum. The crude solid residue, L8,
was purified by HPLC using a 85/15 water/acetonitrile (0.1% TFA in
each) flow rate 4 mL, Rt, 10.2 min. 1H NMR (DMSO-d6) δ: 8.15 (m,
1H, HNCO(Lys-linker)), 8.05 (s, 1H, HC-triazole), 7.75 (m, 1H,
HNCO(Lys)), 6.34 (m, 2H, NH(CO)NH), 4.40−3.99 (m, 7H, H2N-
triazole, H2C−N(triazole), HC(NHCO2(Glu), HC(NHCO2(Lys),
HC(NH2)(triazole)), 3.21 (m, 2H, H2CCH(triazole)), 3.03 (m, 2H,
H2CNH(Lys)), 2.40−2.09 (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)). 13C NMR (DMSO-d6) δ: 175.00
(CO2H), 174.64 (CO2H), 174.22 (CO2H), 172.87 (CO2H), 172.42
(CONH), 170.44 (CONH), 157.77 (NHCONH), 140.88 (C-
triazole), 124.04 (C−H, triazole), 52.73 (CH, Glu), 52.24, 52.11
(CH, Lys), (CH, Lys), 51.91 (CHNH2), 49.59 (CH, NCH2(triazole)),
44.16 (CH2NH), 38.75 (CH2NH, Lys-linker), 35.87 (CH2CO, linker),
35.48 (CH2CO, linker), 32.24 (CH2CH(triazole)), 30.87 (CH2CO,
Glu)), 30.35 (CH2CH, Glu)), 29.78, 29.32, 28.96, 28.86, 28.75, 27.97,
26.57, 25.71 (CH2(linker), (Lys), (Lys-linker)) 23.09, 22.92, 22.66
(CH2(Lys)),(CH2(Lys-linker)). ESMS: 743 [M + H]+. HRESI+-MS:
calcd for C31H50N8O13, 743.3576 [M + H]+; found, 743.3570 (found).
(3S,7S)-26-(4-(2-Amino-2-carboxyethyl)-1H-1,2,3-triazol-1-
yl)-5,13,20-trioxo-4,6,12,21-tetraazahexacosane-1,3,7,22-tet-
racarboxylic Acid Tricarbonyl Rhenium(I), ReL8. L8 (0.050g, 0.67
mmol) was dissolved in 2 mL of water. A solution of [Re-
(CO)3(H2O)3]Br23 (0.029g, 0.67 mmol in 0.5 mL methanol) was
added, and the reaction mixture was refluxed for 4 h. The crude solid,
compound ReL8, was purified by HPLC using a gradient HPLC for 40
min, flow rate 8 mL/min, 80/10 water/acetonitrile (0.1% TFA in
each) to 70/10 water/acetonitrile (0.1% TFA in each) water flow rate
8 mL, Rt, 9.8 min. 1H NMR (DMSO-d6) δ: 8.24 (m, 1H, HNCO(Lys-
linker)), 8.05 (m, 1H, HC-triazole), 7.75 (m, 1H, HNCO(Lys)), 6.34
(m, 2H, NH(CO)NH), 5.96 (m, 1H, H2N-triazole), 5.25 (m, 1H,
H2N-triazole), 4.45−3.85 (m, 5H, H2C−N(triazole), HC-
(NHCO2(Glu), HC(NHCO2(Lys), HC(NH2)(triazole)), 3.25 (m,
1H, H2CCH(triazole)), 2.98 (m, 3H, H2CCH(triazole), H2CNH-
(Lys)), 2.31−2.01 (m, 6H, H2CCO2(Glu), H2CCO2(linker),
H2CCO2(linker)), 2.01−1.54 (m, 6H, H2CCH(Glu), H2CCH(Lys-
linker), H2CCH(Lys)), 1.54−1.22 (m, 16H, (CH2)2 (Lys), (CH2)2
(Lys-Linker), (CH2)4 (linker)). ESMS: 1013 [M + H]+. HRESI+-MS:
calcd for C34H50N8O16Re, 1013.2898 [M + H]+; found, 1013.28986.
2-[3-(1-Carboxy-5-(7-[1-carboxy-5-(carboxymethyl-pyridin-
2-ylmethyl-amino)-pentylcarbamoyl]-heptanoylamino)-pen-
tyl)-ureido]-pentanedioic Acid, L9. Compound L9 was prepared
following Scheme 2 in a total of six steps as described below.
Compound 3 was synthesized using commercially available Fmoc-
Lys(Boc)-OH and 4-methoxy-benzylchloride in presence of cesium
carbonate in DMF at rt.16 Selective removal of Boc from 3 was
performed using p-toluenesulfonic acid.16 Briefly, a solution of 3 (2 g,
3.40 mmol in 20 mL EtOAc) was cooled to 0−2 °C in an ice bath, and
p-toluenesulfonic acid monohydrate (0.64 g, 2.60 mmol) in 5 mL of
absolute ethanol was added. The cooling bath was removed, and the
reaction mixture was allowed to warm to room temperature for 2 h.
The reaction mixture was then concentrated to a thick oil under
reduced pressure, and the mixture was purified by flash chromatog-
raphy using 5/95 MeOH/CH2Cl2 to afford product 4 as colorless solid
in 55% (1.24 g, 1.87 mmol) yield. ESIMS m/z: 660 [M]+. A solution
of 4 (0.66 g, 1 mmol, in 40 mL CH2Cl2) was neutralized with TEA
(140 μL, 1.5 mmol) and was cooled at 4 °C by ice-bath. A solution of
pyridine-2-aldehyde (0.086g, 0.8 mmol) was added slowly to the ice
cold solution of 4 for a period of 30 min and was stirred for another 30
min. Then ice-bath was removed and the solution was stirred at rt for
2 h. Sodium triacetoxyborohydride (0.43 g, 2 mmol) was added
portionwise to the reaction mixture followed by acetic acid (126 μL, 2
Considerations for clinical translation of one or more of the
radioligands studied herein, or other compounds of this class,
include: (1) high concentration within tumor (% ID/g), (2)
high binding specificity (PSMA+ PC3 PIP to PSMA− PC3 flu
uptake ratio), (3) longer retention in PSMA+ PC3 PIP vs
kidney, and (4) low liver and gastrointestinal uptake. While
difficult to optimize for all of these parameters concurrently, the
compounds synthesized in this small series could be ranked
with respect to their desirability for translation. Because all 12
99mTc-labeled compounds demonstrated high tumor uptake
(>20% ID/g at 2 h postinjection), we next compared the values
in PSMA+ PC3 PIP to PSMA− PC3 flu tumors. Compound
[
99mTc]L8 proved superior in this regard, as well as with respect
to PSMA+ PC3 PIP tumor-to-blood, tumor-to-kidney, and
tumor-to muscle ratios. Despite high tumor uptake, other
compounds had either confounding gastrointestinal or spleen
uptake, difficult to predict based on charge and lipophilicity.
Overall, [99mTc]L8 demonstrated characteristics most consis-
tent with a clinically viable agent among both the tricarbonyl
and 99mTc-oxo species.
CONCLUSIONS
■
We have synthesized 12 new low-molecular-weight, urea-based,
99mTc-labeled, PSMA-targeted radiotracers, which differ in the
chelating agent and, in some cases, in lipophilicity by
manipulation of the linker moiety. We found that except for
HYNIC-labeled [99mTc]L19, all radioligands demonstrated
high tumor uptake and retention with the choice of chelator
having a profound effect on pharmacokinetics. Compound
[
99mTc]L8 demonstrated improved tumor uptake and bio-
distribution characteristics compared to all the newly
synthesized and previously reported 99mTc agents from our
laboratory and is likely the most suitable for clinical
development. These results indicate that consideration of the
chelating agent is critical and must be carefully considered
when beginning a new campaign to find the most clinically
viable 99mTc-labeled agent, for compounds of this class and
likely for those intended for other biomedically important
targets.
EXPERIMENTAL SECTION
■
A detailed description of general experimental methods is included in
the Supporting Information.The purity of tested compounds as
determined by analytical HPLC with absorbance at 220 nm was >95%.
(3S,7S,22S)-26-(4-(2-Amino-2-carboxyethyl)-1H-1,2,3-tria-
zol-1-yl)-5,13,20-trioxo-4,6,12,21-tetraazahexacosane-
1,3,7,22-tetracarboxylic Acid, L8. To a solution of Fmoc-Lys(N3)-
OH (0.25 g, 0.63 mmol in 8 mL of 2/1 t-BuOH/H2O), was added
Boc-L-propargylglycine·DCHA (0.25 g, 0.63 mmol in 2 mL of 2/1 t-
BuOH/H2O), followed by sodium ascorbate (4.75 mg, 0.024 mmol in
1 mL water) and Cu(OAc)2·H2O (2 mg, 0.012 mmol in 1 mL water)
and the mixture stirred at room temperature for 12 h under N2 at 30
°C. Quadrapure-IDA resin (20 mg) was added to the solution and was
gently shaked using mechanical shaker at rt for 2 h to remove Cu(II).
The solution was filtered through a Celite bed, and the light-yellow
filtrate was evaporated to dryness. The solid residue was thus obtained
was partitioned between EtOAc and water. The aqueous phases were
re-extracted with EtOAc. The organic phases were combined, dried
over Na2SO4, and evaporated. The crude product was used without
6116
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mmol) and left for 16 h. Solvent was removed under vacuum, and the
residue was dissolved in dichloromethane and extracted with 3 × 50
mL of water. Organic layer was dried under vacuum and purified via a
silica gel column using 4−5% methanol/CH2Cl2 as an eluent to obtain
compound 5 in ∼40%, 0.232 g (0.4 mmol) yield. ESI-MS: 580 [M +
1]+. A solution of 5 (0.1 g, 0.17 mmol in 20 mL DMF) was cooled to 4
°C. To this was added t-butyl bromoacetate (32 μL,0.17 mmol),
potassium iodide (0.028g, 0.17 mmol), and diisopropyl ethylamine (30
μL, 0.17 mmol), respectively, and the mixture stirred for 2 h. The ice-
bath was removed, and the mixture was stirred for 2 days at rt. The
solvent was removed under vacuum, and the residue was dissolved in
50 mL of ethyl acetate and washed with 3 × 50 mL water. The organic
layer was concentrated under vacuum and purified via a silica gel
column using 20/80−30/70 EtOAc/CH2Cl2 as eluent. Thus, 6 was
obtained in 33% (0.039 g) yield. Compound 6 was treated with 4 mL
1/1 TFA/CH2Cl2 at rt for 2 h to simultaneously remove Boc and t-
butyl groups. After solvent removal, crude solid was lyophilized and
treated with 2 mL of 20/80 piperidine/DMF at rt for 20 min. Solvent
was removed under vacuum. The colorless crude material was
dissolved in water (20 mL) and extracted with ethyl acetate (3 × 10
mL). Compound 7 was obtained from the water layer in 48% yield.
Synthesis of L11−L18. These compounds were prepared
following a combined solid phase and solution phase synthetic
strategy as described for L11.
2-(3-[1-Carboxy-5-(7-(5-carboxy-5-[3-phenyl-2-(3-phenyl-2-{2-[2-
(2-tritylsulfanyl-acetylamino)-acetylamino]-acetylamino)-propio-
nylamino)-propionylamino]-pentylcarbamoyl)-heptanoylamino)-
pentyl]-ureido)-pentanedioic Acid, L11. Lys(Boc)-Wang resin (250
mg, 0.43 mmol) 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, followed by gently shaking 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.50 After swelling the resin in DMF, a
solution of Fmoc-Gly-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). The
coupling efficiency was assessed by the Kaiser test. The aforemen-
tioned sequence was repeated for two more coupling steps with Fmoc-
Gly-OH. Final compound was cleaved from the resin using TFA/
CH2Cl2/TES (1:1:0.02) and concentrated under vacuum to produce
9. The concentrated product was purified by using a C18 SepPak Vac
10g column. The product was eluted with a solution 70/30 water/
acetonitrile (0.1% TFA in each). ESIMS: 482 [M]+. Lyophilized 9
(100 mg, 207 μmol in 5 mL of DMF) was added to 2 (120 mg, 207
μmol in 4 mL DMF) followed by DIEA (428 μmol, 60 μL) and then
stirred at rt for 2 h. After solvent removal, the colorless solid residue
thus obtained was purified by solid phase extraction by using a C18
SepPak Vac 10g column using an eluent of 60/40 water/acetonitrile
(0.1% TFA in each). ESIMS: 941 [M + H]+. Yield: ∼70 mg, (36%).
The isolated compound (70 mg, 74 μmol) was treated with 20/80
piperdine/DMF solution (5 mL × 2) to remove Fmoc group. Some
colorless precipitate was appeared. Water (0.1 mL) was added to the
reaction mixture to dissolve the precipitate and left at rt for 5 min and
was purified by solid phase extraction (C18 SepPak Vac 5g column
using an eluent of 85/15 water/acetonitrile (0.1% TFA in each)). ESI-
MS: 717 [M]+. Yield: ∼35 mg (∼66%). To a solution of NHS-ester of
mercaptoacetic acid24 (26 mg, 60 μmol in 3 mL of DMF) was added
the above compound (36 mg, 50 μmol in 2 mL of DMF) for 2h at rt.
After solvent removal product was purified by preparative RP-HPLC
to obtain L11 in ∼20 mg (32%). L11−15 purified by same general
HPLC method. The solvent system is 0−5 min 60% A and 40% B, 5−
25 min 60−50% A and 40−50% B, 25−35 min 50−60% A and 50−
1
Compound 7 was further purified via C18 solid phase extraction. H
NMR (MeOD) for 7: δ 8.25 (d, 1H), 7.82 (t, 1H), 7.33 (t, 1H), 6.65
(d, 2H), 4.2 (m, 1H) 3.82 (s, 2H), 3.20 (s, 2H), 2.85 (t, 2H), 2.62 (t,
2H), 2.75−2.39 (m, 6H). ESI-MS: 295 [M + 1]+. A solution of 216
(0.020 g, 0.0.034 mmol in 100 μL of DMF) was added to a stirred
solution of 5 (0.010 g, 0.034 mmol in 100 μL DMF) at rt followed by
the addition of DIEA (0.05 μL, 0.034 mmol). The reaction mixture
was left for 3 h at room temperature. The reaction mixture was then
concentrated under reduced pressure dissolved in 1 mL of water and
purified by HPLC using a isocratic method of 80/20 water (0.1%
TFA)/acetonitrile (0.1% TFA) as mobile phase, flow rate 4 mL/min;
Rt, 14 min. 1H NMR (DMSO-d6): δ 8.24 (m, 1H, HNCO(Lys-
linker)), 7.75 (m, 1H, HNCO(Lys)), 8.32 (t, 1H, Py), 7.85 (m, 2H,
Py), 7.33 (t, 1H), 6.31 (m, 2H, NH(CO)NH), 4.32−4.18 (m, 3H,
HC(NHCO2(Glu)), HC(NHCO2(Lys), HC(NHCO2(Lys)), 4.14 (m,
2H, PyCH2)), 3.42 (m, 2H, CH2CO2), 2.95 (m, 4H, H2CNH(Lys),
H2CNH(Lys-linker)), 2.32−2.02 (m, 6H, H2CCO2(Glu),
H2CCO2(linker), H2CCO2(linker)), 2.01−1.56 (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)). ESIMS m/z:
753 [M + H]+. HRFAB+-MS: calcd for C34H53N6O13 [M + H]+,
753.3671; found, 753.3675.
1
40% B, Flow rate 4 mL/min, Rt, 11.2 min. H NMR (DMSO-d6): δ
8.23−7.76 (m, 5H, HNCO(Lys-linker), HNCO(Lys), HNCO(Gly)),
7.42−7.20 (m, 15H, trityl), 6.34 (m, 2H, NH(CO)NH), 4.33−3.98
(m, 7H, HC(NHCO2(Glu), HC(NHCO2(Lys), HC(NHCO2(Lys),
H2CNH(Gly)(2)), 3.54 (m, 2H, H2C−S-trityl), 3.0−2.95 (4H,
H2CNH(Lys), H2CNH(Lys)), 2.32−2.02 (m, 6H, H2CCO2(Glu),
H2CCO2(linker), H2CCO2(linker)), 2.05−1.53 (m, 6H, H2CCH-
(Glu), H2CCH(Lys-linker), H2CCH(Lys)), 1.53−1.22 (m, 16H,
(CH2)2 (Lys), (CH2)2 (Lys-linker), (CH2)4 (linker)). HRESI+-MS:
calcd for C51H68N7O14S, 1034.4545 [M + H]+; found, 1034.4542.
12-Benzyl-4,7,10,13,21,28,36-heptaoxo-1,1,1-triphenyl-2-thia-
5,8,11,14,20,29,35,37-octaazatetracontane-15,34,38,40-tetracar-
boxylic Acid, L12. MAG-2 chelator, amino acid residues are Lys, Gly,
2-{3-[5-(7-(5-[Bis-(1H-imidazol-2-ylmethyl)-amino]-1-car-
boxy-pentylcarbamoyl)-heptanoylamino)-1-carboxy-pentyl]-
ureido}-pentanedioic Acid, L10. The bisimidazolyl lysine chelate,
8, was prepared following a literature procedure.16 Briefly, Fmoc-Lys-
OH was reacted with imidazole-2 aldehyde in the presence of
NaBH(OAc)3 and acetic acid in dichoroethane. Solution was stirred
for 20 h. The product was diluted with CH2Cl2 and washed with water.
The solid residue obtained after evaporation of the organic layer was
further purified by SiO2 column chromatography using 20/80 MeOH/
CH2Cl2 as eluant. Fmoc removal of the compound was performed
using a 20/80 piperidine/DMF solution. After solvent removal,
compound was purified using a C18 solid phase extraction using 90/10
H2O/CH3CN as eluant. ESI-MS 307 [M + H]+. Compound 8 (10 mg,
0.03 mmol in 1 mL DMF) was added to a solution of 2 (28 mg, 0.06
mmol) and DIEA (50 μL) and left for 2 h at rt. After solvent
evaporation, product was purified using a C18 column. The compound
was further purified by HPLC using same method as L8, Rt, 13.5 min.
1H NMR (DMSO-d6): δ 8.24 (m, 1H, HNCO(Lys-linker)), 7.75 (m,
1H, HNCO(Lys)), 7.21 (ds, 2H, Im), 7.06 (ds, 2H, Im), 6.34 (m, 2H,
NH(CO)NH), 4.33−4.10 (m, 7H, ImCH2, HC(NHCO2(Glu),
HC(NHCO2(Lys), HC(NHCO2(Lys)), 3.0−2.95 (4H, H2CNH(Lys),
H2CNH(Lys)), 2.32−2.02 (m, 6H, H2CCO2(Glu), H2CCO2(linker),
H2CCO2(linker)), 2.01−1.55 (m, 6H, H2CCH(Glu), H2CCH(Lys-
linker), H2CCH(Lys)), 1.55−1.20 (m, 16H, (CH2)2 (Lys), (CH2)2
(Lys-Linker), (CH2)4 (linker)). HRESI+-MS: calcd for
C34H53N9NaO11, 786.3762 [M + Na]+; found, 786.3757(found).
1
Gly, Phe. Rt, 16 min. H NMR (DMSO-d6): δ 8.23−7.76 (m, 6H,
HNCO(Lys-linker), HNCO(Lys), HNCO(Gly), HNCOPhe)), 7.42−
7.20 (m, 20H, trityl, Ph), 6.34 (m, 2H, NH(CO)NH), 4.33−3.98 (m,
8H, HC(NHCO2(Glu), HC(NHCO2(Lys), HC(NHCO2(Lys),
HCNH(Phe), H2CNH(Gly)(2)), 3.54−3.44 (m, 4H, H2C−S-trityl,
H2C(Phe)), 3.0−2.95 (4H, H2CNH(Lys), H2CNH(Lys)), 2.32−2.06
(m, 6H, H2CCO2(Glu), H2CCO2(linker), H2CCO2(linker)), 2.02−
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)). HRESI+-MS: calcd for C60H77N8O15S, 1180.5151 [M +
H]+; found, 1180.5154.
12,15-Dibenzyl-4,7,10,13,16,24,31,39-octaoxo-1,1,1-triphenyl-2-
thia-5,8,11,14,17,23,32,38,40-nonaazatritetracontane-18,37,41,43-
tetracarboxylic Acid, L13. MAG-2 chelator, amino acid residues are
1
Lys, Gly, Gly, Phe, Phe. Rt, 22 min. H NMR (DMSO-d6): δ 8.23−
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7.76 (m, 7H, HNCO(Lys-linker), HNCO(Lys), HNCO(Gly),
HNCOPhe), 7.42−7.20 (m, 25H, trityl, Ph), 6.34 (m, 2H,
NH(CO)NH), 4.33−3.98 (m, 9H, HC(NHCO2(Glu), HC-
(NHCO2(Lys), HC(NHCO2(Lys), HCNH(Phe), H2CNH(Gly)(2)),
3.54−3.41 (m, 6H, H2C−S-trityl, H2C(Phe) (2)), 3.0−2.95 (4H,
H2CNH(Lys), H2CNH(Lys)), 2.32−2.03 (m, 6H, H2CCO2(Glu),
H2CCO2(linker), H2CCO2(linker)), 2.02−1.56 (m, 6H, H2CCH-
(Glu), H2CCH(Lys-linker), H2CCH(Lys)), 1.55−1.20 (m, 16H,
(CH2)2 (Lys), (CH2)2 (Lys-Linker), (CH2)4 (linker)). HRESI+-MS:
calcd for C69H86N9O16S, 1328.5913 [M + H]+; found, 1328.5917.
4,7,10,13,21,28,36-Heptaoxo-1,1,1-triphenyl-2-thia-
5,8,11,14,29,35,37-heptaazatetracontane-15,34,38,40-tetracarbox-
ylic Acid, L14. MAG-3 chelator, amino acid residues are Lys, Gly, Gly,
Gly. Rt, 13 min. 1H NMR (DMSO-d6): δ 8.23−7.76 (m, 6H,
HNCO(Lys-linker), HNCO(Lys), HNCO(Gly)), 7.42−7.20 (m,
15H, trityl), 6.34 (m, 2H, NH(CO)NH), 4.33−3.98 (m, 9H,
HC(NHCO2(Glu)), HC(NHCO2(Lys)), HC(NHCO2(Lys)),
HCNH(Gly)(3)), 3.54 (m, 2H, H2C−S-trityl), 3.0−2.95 (4H,
H2CNH(Lys), H2CNH(Lys)), 2.32−2.02 (m, 6H, H2CCO2(Glu),
H2CCO2(linker), H2CCO2(linker)), 2.0−1.55 (m, 6H, H2CCH(Glu),
H2CCH(Lys-linker), H2CCH(Lys)), 1.54−1.23 (m, 16H, (CH2)2
(Lys), (CH2)2 (Lys-linker), (CH2)4 (linker)). HRESI+-MS: calcd for
C54H72N7O15S, 1090.4907 [M + H]+; found, 1090.4910.
2.99 (m, 4H, H2CNH(Lys), H2CNH(Lys)), 2.27−2.20 (m, 2H,
H2CCH(Glu)), 2.20−2.00 (m, 4H, m, 4H, H2CCO2(linker),
H2CCO2(linker)), 1.92−1.62 (m, 6H, H2CCH(Glu), H2CCH(Lys-
linker)), 1.52−1.26 (m, 16H, (CH2)2 (Lys), (CH2)2 (Lys-linker),
(CH2)4 (linker)). ESIMS 878 [M]+. HRESI+-MS: calcd for
C35H59N8O16S, 879.3770; found, 879.3770.
5-Amino-32-(carboxymethyl)-29-(mercaptomethyl)-
5,13,20,28,31,34-hexaoxo-4,6,12,21,27,30,33-heptaazahexatria-
contane-1,3,7,26,36-pentacarboxylic Acid, L18. Amino acid residues
are Lys, Cys, Asp, Asp. Rt, 18 min, (HPLC method same as L16)
8.23−7.97 (m, 5H, HNCO(Lys-linker), HNCO(Lys), NHCO(Cys)
NHCO (Asp)), 6.35−6.30 (m, 2H, NHCONH), 4.81−3.89 (m, 6H,
HCNH(Cys), HCNH(Asp), HC(NHCO2(Glu), HC(NHCO2(Lys)),
HC(NHCO2(Lys-linker))), 3.65−3.02 (m, 6H, H2CSH, H2CCO2
(Asp)), 3.02−2.99 (m, 4H, H2CNH(Lys), H2CNH(Lys)), 2.27−
2.20 (m, 2H, H2CCH(Glu)), 2.20−2.00 (m, 4H, H2CCO2(linker),
H2CCO2(linker)), 1.92−1.62 (m, 6H, H2CCH(Glu), H2CCH(Lys-
linker)), 1.53−1.26 (m, 16H, (CH2)2 (Lys), (CH2)2 (Lys-Linker),
(CH2)4 (linker)). HRESI+-MS: calcd for C37H60N8O18S, 936.3746
[M]+; found, 936.3748.
2-(3-[1-Carboxy-5-(7-(1-carboxy-5-[(6-hydrazino-pyridine-3-
carbonyl)-amino]-pentylcarbamoyl)-heptanoylamino)-pentyl]-
ureido)-pentanedioic Acid, L19. To a solution of 1025 (10 mg, 16
μmol, 0.5 mL DMSO) was added HYNIC-OSu (12 mg, 32 μmol in
0.5 mL DMSO) and DIEA (50 μL) and left at rt for 2hr. After solvent
evaporation, the compound was purified using 2g C18 cartridge. The
product was lyophilyzed. Compound was treated 1/1 of 2 mL of TFA/
CH2Cl2 for 2 h at rt. After solvent evaporation, the solid residue was
purified by HPLC (method same as L8), Rt, 12.5 min. 1H NMR
(DMSO-d6): 8.35 (s, 1H, Py), 8.23 (m,1H, HNCO(Lys-linker)), 7.75
(m, 2H, HNCO(Lys), CHPy), 7.25 (m, 1H, CHPy), 6.35−6.30 (m,
2H, NHCONH), 4.13−3.92 (m, 3H, HC(NHCO2(Glu), HC-
(NHCO2(Lys), HC(NHCO2(Lys-linker)), 3.01−2.98 (m, 2H,
H2CNH(Lys)), 2.27−2.20 (m, 2H, H2CCO2(Glu)), 2.22−2.02 (m,
4H, H2CCO2(linker), H2CCO2(linker)), 2.09−1.65 (m, 6H, H2CCH-
(Glu), H2CCH(Lys-linker), 1.52−1.23 (m, 16H, (CH2)2 (Lys),
(CH2)2 (Lys-Linker), (CH2)4 (linker)). HRESI+-MS: calcd for
C32H50N8O12, 738.3548 [M + H]; found, 738.3543(found).
Radiochemistry. All radiolabeled compounds were purified by
HPLC using a Phenomenex C18 Luna 10 × 250 mm2 10 μm column.
HPLC was performed on a Varian Prostar system (Palo Alto, CA),
equipped with a Varian Prostar 325 variable wavelength UV detector
and a Bioscan Flow-Count in-line HPLC radioactivity detector. All
final compounds were obtained in >98% purity, as determined by
HPLC (Supporting Information). The following HPLC solvent
methods were used to purify the radiolabeled ligands [99mTc]L8−
L19 from the unlabeled ligand. HPLC was performed using solvent A
(0.1% TFA in water) and solvent B (0.1% TFA in CH3CN), flow rate
4 mL/min. Method 1: gradient method, 0−15 min 90−80% A and
10−20% B, 15−35 min 80−65% A and 20−35% B, 35−45 min 65−
90% A and 35−10% B. Method 2: isocratic method, the mobile phase
was 80% solvent A and 20% solvent B. Method 3: gradient method,
0−5 min 85% A and 15% B, 5−25 min 85−50% A and 15−50% B,
25−35 min 50−20% A and 50−80% B, 35−40 min 20−85% A and
80−15% B. Method 4: gradient method, 0−5 min 80% A and 20% B,
5−25 min 80−40% A and 20−60% B, 25−35 min 40−80% A and 60−
20% B. Method 5: isocratic method, 86% solvent A and 14% solvent B.
Retention time of the compounds are listed in Supporting Information
Table S1. HPLC chromatograms of [99mTc]L8−L19 and stability
studies of [99mTc]L8 and [99mTc]L15 are reported in the Supporting
Information.
(19S,34S,38S)-6,9,12-Tris(hydroxymethyl)-4,7,10,13,21,28,36-hep-
taoxo-1,1,1-triphenyl-2-thia-5,8,11,14,20,29,35,37-octaazatetra-
contane-19,34,38,40-tetracarboxylic Acid, L15. MAS-3 chelator,
amino acid residues are Lys, Ser, Ser, Ser. Rt, 9 min. 1H NMR
(DMSO-d6): δ 8.23−7.76 (m, 6H, HNCO(Lys-linker), HNCO(Lys),
HNCO(Ser)), 7.42−7.20 (m, 15H, trityl), 6.34 (m, 2H, NH(CO)-
NH), 4.63−3.98 (m, 12H, HCNH(Ser)(3), HC(NHCO2(Glu),
HC(NHCO2(Lys)), HC(NHCO2(Lys-linker), H2CCO2((Ser)(3))),
3.67 (sb, 3H, HOCH2), 3.54 (m, 2H, H2C−S-trityl), 3.0−2.95 (4H,
H2CNH(Lys), H2CNH(Lys)), 2.32−2.03 (m, 6H, H2CCO2(Glu),
H2CCO2(linker), H2CCO2(linker)), 2.02−1.56 (m, 6H, H2CCH-
(Glu), H2CCH(Lys-linker), H2CCH(Lys)), 1.55−1.23 (m, 16H,
(CH2)2 (Lys), (CH2)2 (Lys-linker), (CH2)4 (linker)). HRESI+-MS:
calcd for C56H77N8O18S2, 1181.5077 [M + H]+; found, 1181.50781.
(10R,29S,33S)-4,8,16,23,31-Pentaoxo-1,1,1-triphenyl-7-(2-
(tritylthio)acetamido)-2-thia-5,9,15,24,30,32-hexaazapentatriacon-
tane-10,29,33,35-tetracarboxylic Acid, L16. Diamide dithiol
(DADT) chelating agent, amino acid residues are Lys and 1,3-
diaminopropionic acid. Rt, 49 min, the HPLC method is 0−10 min
70% A and 30% B, 10−60 min 70−10% A and 30−90% B, 60−65 min
1
10−70% A and 90−30% B, Flow rate 8 mL/min. H NMR (DMSO-
d6): δ 8.25−7.98 (m, 5H, HNCO(Lys-linker), HNCO(Lys), NHCO-
(Dap)), 7.45−7.14 (m, 30H, trityl), 6.37−6.33 (m, 2H, NHCONH),
5.10 (m, 1H, CHNH, dap), 4.33−4.20 (m, 3H, HC(NHCO2(Glu),
HC(NHCO2(Lys)), HC(NHCO2(Lys-linker)), 3.54−3.34 (m, 6H,
H2C−S-trityl, H2CNH2(dap)), 3.02−2.99 (m, 4H, H2CNH(Lys),
H2CNH(Lys)), 2.27−2.20 (m, 2H, H2CCH(Glu)), 2.20−2.00 (m,
4H, H2CCH(Lys-linker), H2CCH(Lys)), 1.92−1.62 (m, 6H, H2CCH-
(Glu), H2CCH(Lys-linker)), 1.52−1.26 (m, 16H, (CH2)2 (Lys),
(CH2)2 (Lys-linker), (CH2)4 (linker)). HRESI+-MS: calcd for
C71H84N7O14S2, 1322.5518 [M + H]+; found, 1322.5520.
2-(3-19-1-Carboxy-pentylcarbamoyl)-heptanoylamino]-1-car-
boxy-pentyl}-ureido)-pentanedioic Acid, L17. Amino acid residues
are Lys, Cys, Asp, Gly. Rt, 38 min. Compound 12 (10 mg, 15.5 μmol
in 0.5 mL) was mixed with 2 (10 mg, 7.4 μmol, 17.4 in 1 mL DMF)
and left at rt for 2 h. Solvent was reduced under vacuum, and residue
showed the expected mass 1100 [M + H]+. The residue was dissolved
in 1 mL of 20/80 piperidine/DMF. Some colorless precipitate was
appeared. Water (0.1 mL) was added to the reaction mixture to
dissolve the precipitate and left at rt for 5 min. The solution was
evaporated to dryness, and the residue was dissolved in water and
purified by HPLC to isolate L17 using the same method as L16. Rt, 20
min. 1H NMR (DMSO-d6): δ 8.22−7.86 (m, 5H, HNCO(Lys-linker),
HNCO(Lys), NHCO(Cys) NHCO (Asp)), 6.35−6.30 (m, 2H,
NHCONH), 4.81−3.89 (m, 5H, HCNH(Cys), HCNH(Asp), HC-
(NHCO2(Glu), HC(NHCO2(Lys)), HC(NHCO2(Lys-linker)),
3.65−3.02 (m, 6H, H2CNH2(Gly), H2CSH, H2CCO2 (Asp)), 3.02−
Radiolabeling of L8−L10. These compounds were synthesized in
radioactive (99mTc-labeled) form using the same general method as
described previously by us using the Isolink kit.16 All 99mTc-labeled
compounds were synthesized in radiochemical yields of 60−90% and
radiochemical purities of >98%. Briefly, 414.7 MBq (11.2 mCi) in 1
mL of saline Na[99mTcO4] was added to the Isolink kit, and the
reaction mixture was heated in a water bath at 95 °C for 20 min then
allowed to cool to rt to prepare [99mTc(CO)3(H2O)3]+. Briefly, for
99mTc[L8] preparation, 500 μL of the [99mTc(CO)3(H2O)3]+ solution
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(222 MBq/6 mCi)) was neutralized with 100 μL of 1(N) HCl. To this
solution was added a 100 μL of phosphate-buffered saline (PBS)
solution and 100 μL of a solution of L8 (2 mg, 2.7 μmol in 1 mL
water) and was heated at 95 °C for 30 min. This was diluted with 750
μL of the HPLC mobile phase and purified by radio-HPLC. The major
radioactive peak constituting desired product (148 MBq/4 mCi)
eluted at 28 min. Radiochemical yield: 64.5%. Radiochemical purity >
98%, specific activity > 411 GBq/μmol (1.1 × 104 mCi/ μmol). For
GraphPad Prism version 4.00 for Windows (GraphPad Software, San
Diego, California).
Tumor Models. Animal studies were carried out in full compliance
with the regulations of the Johns Hopkins Animal Care and Use
Committee. The 6−8-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 (2 × 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.
SPECT-CT Imaging of PSMA+ PC3 PIP and PSMA− PC3 flu
Xenografts. Compounds [99mTc]L8−L19 were imaged in PSMA+
PC3 PIP and PSMA− PC3 Flu xenograft models. Mice (N = 2) were
injected via the tail vein with approximately 37 MBq (1 mCi) of
radiotracer formulated in 100 μL of saline at pH ∼ 7. Mice were
anesthetized with 3% isofluorane, placed in the scanner, and
maintained at 1% isoflurane throughout the imaging procedure. A
Gamma Medica-Ideas (Northridge, CA) X-SPECT scanner equipped
with two opposing low-energy 0.5 mm aperture pinholes and tunable
CT was used for all scans. Mice were scanned over 180° in 5.5°, 30 s
increments. A CT scan was performed prior to or after scintigraphy for
anatomical coregistration. Data were reconstructed and fused using
commercial software from the vendor (Gamma Medica-Ideas), which
includes a 2D-ordered subsets-expectation maximum (OS-EM)
algorithm. Volume rendered images were generated using Amira
software (http://www.vsg3d.com/amira).
[
99mTc]L8 and [99mTc]L10, method 1 was used. Method 2 was
employed to purify [99mTc]L9.
Radiolabeling of L11−L18. Radiolabeling for ligands L11−L18
were performed following a literature procedure.26 Briefly, compound
L11 (1 mg, 75.3 μmol) was dissolved in 1 mL of 0.5 M ammonium
acetate buffer at pH 8. Disodium tartrate dihydrate was dissolved in
the labeling buffer of 0.5 M ammonium acetate (pH 8.5) to a
concentration of 50 mg/mL. Ascorbic acid−HCl solution was
prepared by dissolving ascorbic acid in 10 mM HCl to a concentration
of 3.0 mg/mL. A fresh 4 mg/mL SnCl2 was prepared using ascorbic−
HCl solution. A solution of L11 (80 μL) was combined to a solution
of 50 μL of 0.25 M ammonium acetate and 20 μL of tartrate buffer.
This solution was purged with N2 for 2 min. To this solution was
added the SnCl2 solution (10 μL) ,and pH of the solution was adjusted
∼8−8.5. 99mTc-pertechnetate 10−20 mCi in 200 μL was added to the
solution and was heated ∼90−100 °C (boiling water bath) for 20 min.
Reaction mixture was cooled, diluted with 850 μL of water, and
purified by HPLC method 3. Two radiolabeled products were isolated
for L11−L13 and designated as [99mTc]L11A−L13A (45%) and
[
99mTc]L11B-L13B (55%) with an overall yield ∼70−90% without
In Vivo Binding Specificity. To validate further the specificity of
compounds in vivo, mice bearing PSMA+ PC3 PIP and PSMA− PC3
flu tumors were treated with 1 mg of the high-affinity PSMA inhibitor,
2N-[[[(1S)-1-carboxy-3-methylbutyl]amino]carbonyl]-L-glutamic acid,
(ZJ43), in 100 μL of saline 30 min prior to the injection of [99mTc]
L11, ∼37 MBq (1 mCi in 100 μL of saline).28 SPECT-CT imaging
commenced at 30 min after administration of ZJ43, the PSMA
blocking agent.
decay correction. To get a good separation between two radiolabeled
products for [99mTc]L11−L13A and B, method 4 was used.
Radiolabeling of L19. Tricine was used as coligand for HYNIC
labeling. Radiolabeling for ligands L19 was performed following a
literature procedure.51 Briefly, to 50 μL of HYNIC ligand (2 mg/mL)
was added 50 μL of 0.5 M NaHCO3 and 100 μL of tricine (7 mg/mL
in water), and the solution was purged with N2 for 2 min. Na99mTcO4
(185 mBq/5 mCi in 200 μL saline) and 10 μL of freshly prepared
SnCl2 (1 mg/mL in 10 mM HCl) were added to the solution and left
at rt for 30 min. HPLC method 5 was used to isolate [99mTc]L19.
Radiolabeled product was obtained in ∼60% yield and ∼95% purity.
Formulation. All radiolabled products were neutralized with ∼20−
30 μL of 1 M sodium bicarbonate and evaporated to dryness under
vacuum. The obtained solid residues was dissolved in 200 μL of saline
and used for imaging and biodistribution studies after appropriate
dilution with saline.
Cell Lines. Sublines of the androgen independent PC3 human PCa
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). All cell cultures were maintained in an atmosphere containing 5%
carbon dioxide (CO2) at 37.0 °C in a humidified incubator.
NAALADase Assay. The PSMA inhibitory activity of L8−L19 and
ReL8 were determined using a fluorescence-based assay according to a
previously reported procedure.22 Briefly, lysates of LNCaP cell extracts
(25 μL) were incubated with the inhibitor (12.5 μL) in the presence of
4 μM N-acetylaspartylglutamate (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.52 Assays
were performed in triplicate. Data analysis was performed using
Biodistribution. Mice bearing PSMA+ PC3 PIP and PSMA− PC3
flu xenografts were injected via the tail vein with 1.85 MBq (50 μCi) of
[
99mTc]L8, [99mTc]L11, [99mTc]L13−L15, or [99mTc]L18 (N = 4 per
study). At 30 min and 1, 2, and 5 h postinjection, mice were sacrificed
after which heart, lungs, liver, stomach, pancreas, spleen, fat, kidney,
muscle, small and large intestines, urinary bladder, PSMA+ PC3 PIP
and PSMA− PC3 flu tumors, and a 0.1−0.2 mL aliquot of blood were
collected. Each organ was weighed, and the tissue radioactivity was
measured with an automated gamma counter (1282 Compugamma
CS, Pharmacia/LKBNuclear, Inc., Gaithersburg, MD). The percentage
of injected dose per gram of tissue (% ID/g) was calculated by
comparison with samples of a standard dilution of the initial dose. All
measurements were corrected for decay.
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 general experimental methods, spectral data and
supporting SPECT-CT imaging figures. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*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, MD 21287. 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, MD 21287.
6119
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Article
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful for the following sources of support: K25
CA148901, NIH NCI U54CA151838, NCI CA134675. There
are no potential conflicts of interest.
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ABBREVIATIONS USED
■
PSMA, prostate-specific membrane antigen; GCPII, glutamate
carboxypeptidase II; NAALADase, N-acetylated-∝-linked acid-
icdipeptidase; DCFBC, N-[N-[(S)-1,3-dicarboxypropyl]-
carbamoyl]-(S)-4-fluorobenzyl-L-cysteine; PET, positron emis-
sion tomography; SPECT, single photon emission computed
tomography; SAAC, single amino acid chelate; 2-PMPA, 2-
(phosphonomethyl)pentanedioic acid; MAG-2, mercaptoace-
tyldiglycine; MAG-3, mercaptoacetyltriglycine; MAS-3, mer-
captoacetyltriserine; DADT, diamidodithiol and; HYNIC, 2-
hydrazinonicotinic acid
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