SYnthesis and biological evaluation of low molecular weight fluorescent imaging agents for the prostate-specific membrane antigen

Article abstract of DOI:10.1021/bc3003919

Targeted near-infrared (NIR) optical imaging can be used in vivo to detect specific tissues, including malignant cells. A series of NIR fluorescent ligands targeting the prostate-specific membrane antigen (PSMA) was synthesized and each compound was tested for its ability to image PSMA+ tissues in experimental models of prostate cancer. The agents were prepared by conjugating commercially available active esters of NIR dyes, including IRDye800CW, IRDye800RS, Cy5.5, Cy7, or a derivative of indocyanine green (ICG) to the terminal amine group of (S)-2-(3-((S)-5-amino-1-carboxypentyl)ureido)pentanedioic acid 1, (14S,18S)-1-amino-8,16-dioxo-3,6-dioxa-9,15,17-triazaicosane-14,18, 20-tricarboxylic acid 2 and (3S,7S)-26-amino-5,13,20-trioxo-4,6,12,21- tetraazahexacosane-1,3,7,22-tetracarboxylic acid 3. The K_i values for the dye-inhibitor conjugates ranged from 1 to 700 pM. All compounds proved capable of imaging PSMA+ tumors selectively to varying degrees depending on the choice of fluorophore and linker. The highest tumor uptake was observed with IRDye800CW employing a poly(ethylene glycol) or lysine-suberate linker, as in 800CW-2 and 800CW-3, while the highest tumor to nontarget tissue ratios were obtained for Cy7 with these same linkers, as in Cy7-2 and Cy7-3. Compounds 2 and 3 provide useful scaffolds for targeting of PSMA+ tissues in vivo and should be useful for preparing NIR dye conjugates designed specifically for clinical intraoperative optical imaging devices.

Full text of DOI:10.1021/bc3003919

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Article
Synthesis and Biological Evaluation of Low Molecular Weight
Fluorescent Imaging Agents for the Prostate-Specific Membrane Antigen
Ying Chen, Mrudula Pullambhatla, Sangeeta Ray Banerjee, Youngjoo Byun, Marigo
Stathis, Camilo Rojas, Barbara S. Slusher, Ronnie C Mease, and Martin G Pomper
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/bc3003919 • Publication Date (Web): 19 Nov 2012
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Synthesis and Biological Evaluation of Low Molecular Weight Fluorescent Imaging
Agents for the Prostate-Specific Membrane Antigen
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Ying Chen,1 Mrudula Pullambhatla,1 Sangeeta R. Banerjee.1Youngjoo Byun,2 Marigo
Stathis,3 Camilo Rojas,3 Barbara S. Slusher,3 Ronnie C. Mease,1 Martin G. Pomper1
1Russell H. Morgan Department of Radiology, Johns Hopkins Medical School,
Baltimore, MD 21231, USA
2College of Pharmacy, Korea University, 2511 Sejong-ro, Jochiwon-eup, Yeongi-gun,
Chungnam 339-700, South Korea
3NeuroTranslational Drug Discovery Program, Brain Science Institute, Johns Hopkins
Medical School, Baltimore, MD 21231, USA
Corresponding author:
Martin G. Pomper, M.D., Ph.D.
Johns Hopkins Medical School
1550 Orleans Street, 492 CRB II
Phone: 410-955-2789
Fax: 443-817-0990
Email: mpomper@jhmi.edu
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ABSTRACT
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Targeted near-infrared (NIR) optical imaging can be used in vivo to detect specific
tissues, including malignant cells. A series of NIR fluorescent ligands targeting the
prostate-specific membrane antigen (PSMA) was synthesized and each tested for its
ability to image PSMA+ tissues in experimental models of prostate cancer.
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The agents were prepared by conjugating commercially available active esters of NIR
dyes, including IRDye800CW, IRDye800RS, Cy5.5, Cy7 or a derivative of indocyanine
green (ICG) to the terminal amine group of (S)-2-(3-((S)-5-amino-1-
carboxypentyl)ureido)pentanedioic acid 1, (14S,18S)-1-amino-8,16-dioxo-3,6-dioxa-
9,15,17-triazaicosane-14,18,20-tricarboxylic acid 2 and (3S,7S)-26-amino-5,13,20-trioxo-
4,6,12,21-tetraazahexacosane-1,3,7,22-tetracarboxylic acid 3. The Ki values for the dye-
inhibitor conjugates ranged from 1 – 700 pM. All compounds proved capable of imaging
PSMA+ tumors selectively to varying degrees depending on the choice of fluorophore
and linker. The highest tumor uptake was observed with IRDye800CW employing a
polyethylene glycol or lysine-suberate linker, as in 800CW-2 and 800CW-3, while the
highest tumor to non-target tissue ratios were obtained for Cy7 with these same linkers,
as in Cy7-2 and Cy7-3. Compounds 2 and 3 provide useful scaffolds for targeting of
PSMA+ tissues in vivo and should be useful for preparing NIR dye conjugates designed
specifically for clinical intra-operative optical imaging devices.
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INTRODUCTION
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The National Cancer Institute estimates 241,740 new cases and 28,170 new deaths from
prostate cancer (PCa) in 2012.1 PCa is the second leading cause of cancer-related death
in men. Surgery is the most commonly used treatment for clinically localized PCa and
provides a survival advantage compared to watchful waiting.2 A pressing issue in
surgery for PCa is the assurance of a complete resection of the tumor, namely, a negative
surgical margin. A positive surgical margin necessitates adjuvant radiation therapy,
which is required in up to 27.8% of patients3 compared to 9.1% of men undergoing
radical retropubic prostatectomy. Given that currently more than 50,000 radical
prostatectomies are performed annually, these statistics would suggest that more than
9,000 men are undergoing unnecessary adjuvant radiation therapy.4 Given that such
radiation therapy costs in excess of $25,000 per patient, the estimated cost of preventable
positive margins is at least $225M annually.
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Local tumor invasion can be difficult to assess pre- and even intra-operatively, but its
detection during surgery can be augmented through the use of fluorescent dyes that home
to tumor in conjunction with an excitation laser and camera that are tuned to the
excitation and emission wavelengths of the dye employed.5-7 While such intra-operative
tumor detection has been performed using non-targeted agents,8 targeted agents are
coming online.9,10 Because of superior tissue penetration and less interference from the
presence of blood in the surgical field, dyes with emission wavelengths in the near-
infrared (NIR) region of the spectrum (650 – 900 nm) are generally employed for this
purpose.11 Because of its abundant expression on the surface of PCa 12,13 as well as
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within most solid tumor neovasculature,14-17 we have developed a series of NIR-emitting
dyes that target the prostate-specific membrane antigen (PSMA).
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We and others have synthesized a variety of low molecular weight PSMA-targeting
radiotracers to enable imaging of PCa, with several such agents currently in the clinic.18-
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Optical agents have also been synthesized,22-24 and we have previously tested one such
compound in mice to good effect.9 While a variety of radiolabeled PSMA-targeting
antibodies have been used for tumor imaging,25-27 we prefer agents of low molecular
weight due to more tractable pharmacokinetics, namely, more rapid washout from non-
target sites enabling imaging within hours rather than days of administration.
NIR-emitting optical dyes are relatively large organic molecules with extended
conjugation, which presents two potential difficulties for target acquisition in vivo: 1)
steric hindrance when linked to small affinity agents, such as the ureas present in many of
the above mentioned PSMA inhibitors; and, 2) poor pharmacokinetics. Recently, we
reported the preparation and evaluation in vivo of YC-27 (800CW-3), which contains a
lysine-suberate linker between the dye (IRDye800CW, LI-COR Biosciences, Lincoln,
NE) and the PSMA targeting urea.9 With 800CW-3 we were able to visualize a PSMA+
tumor xenograft by optical imaging, demonstrating that with a long lysine-suberate
linking moiety, i.e., a long linker, PSMA inhibitors containing bulky groups will still bind
to the target. Here we extend this work by synthesizing and testing a series of PSMA-
targeted NIR fluorescent agents, each containing either no linker, or one of two different
linking groups that have different pharmacokinetic implications for imaging in vivo.
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EXPERIMENTAL PROCEDURES
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Chemistry. General Methods. All chemicals and solvents were purchased from either
Sigma-Aldrich (Milwaukee, WI) or Fisher Scientific (Pittsburgh, PA). The N-
hydroxysuccinimide (NHS) esters of IRDye800CW and IRDye800RS were purchased
from LI-COR Biosciences. The N-hydroxysulfosuccinimide ester of ICG derivative
(ICG-sulfo-OSu) was purchased from DOJINDO Molecular Technologies (Rockville,
MD). The NHS esters of Cy7 and Cy5.5 were purchased from GE Healthcare
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(Piscataway, NJ). H NMR spectra were obtained on a Bruker Avance 400 MHz
Spectrometer (Billerica, MA). Mass spectra were obtained on a Bruker Esquire 3000
plus system (ESI) or an Applied Biosystems Voyager DE-FTR MALDI-TOF (Foster
City, CA). High-performance liquid chromatography (HPLC) purifications were
performed on a Waters 625 LC system (Milford, MA) or a Varian Prostar System (Varian
Medical Systems, Palo Alto, CA). Optical images were obtained using the Pearl Impulse
Imager (LI-COR Biosciences, Lincoln, NE) and a Xenogen IVIS Spectrum (Caliper Life
Sciences, Hopkinton, MA). HPLC traces of all of the final compounds are available in
the Supplementary Information.
Synthesis of (S)-2-(3-((S)-5-amino-1-carboxypentyl)ureido)pentanedioic acid (1). To
a solution of (10S,14S)-tris(4-methoxybenzyl) 2,2-dimethyl-4,12-dioxo-3-oxa-5,11,13-
triazahexadecane-10,14,16-tricarboxylate 428 (0.264 g, 0.033 mmol) a solution of 3%
anisole in TFA (1 mL) was added and the mixture was reacted at room temperature for 20
min, then concentrated on a rotary evaporator. The crude material was purified by HPLC
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[Econosphere C18, 10 ꢀm, 250 x 10 mm, H2O/CH3CN/TFA (92/8/0.1), 4 mL/min] to
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afford 0.108 g (74%) of 1 (retention time = 4 min ). H NMR (400 MHz, D2O)
δ
4.17-
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4.25 (m, 2H), 2.94-2.98 (m, 2H), 2.46-2.50 (m, 2H), 2.11-2.19 (m, 1H), 1.80-1.98 (m,
2H), 1.61-1.73 (m, 3H), 1.40-1.49 (m, 2H). 13C NMR (400 MHz, D2O): δ 176.9, 176.8,
176.2, 163.0, 162.7, 162.3, 162.0, 158.8, 120.2, 117.3, 114.1, 111.5, 52.7, 52.4, 38.8,
30.0, 29.7, 25.8, 25.7, 21.5. ESI-Mass calcd for C12H22N3O7 [M]+ 320.2, found 320.0.
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Synthesis of 800CW-1. To a solution of 1 (0.5 mg, 1.15 ꢀmol) in DMSO (0.1 mL) was
added N,N-diisopropylethylamine (0.002 mL, 11.5 ꢀmol), followed by the NHS ester of
IRDye800CW (0.3 mg, 0.26 ꢀmol). After 2 h at room temperature the reaction mixture
was purified by HPLC (Econosphere C18, 5ꢀm, 150 × 4.6 mm; mobile phase, A = 0.1%
TFA in H2O, B = 0.1% TFA in CH3CN; gradient, 0 min = 5% B, 5 min = 5% B, 45 min =
100% B; flow rate, 1 mL/min) to afford 0.2 mg (60%) of 800CW-1 (retention time = 15
min). ESI-Mass calcd for C58H74N5O21S4 [M]+ 1304.4, found 1303.8.
Synthesis of 800RS-1 To a solution of 1 (0.2 mg, 0.46 ꢀmol) in DMSO (0.05 mL) was
added N,N-diisopropylethylamine (0.002 mL, 11.5 ꢀmol), followed by the NHS ester of
IRDye800RS (0.2 mg, 0.21 ꢀmol). After 2 h at room temperature the reaction mixture
was purified by HPLC as described for 800CW-1 to afford 0.2 mg (84%) of 800RS-1
(retention time = 23 min). ESI-Mass calcd for C58H73N5O15S2 [M]+ 1143.5, found 572.5
[M+H]2+, 1144.0 [M]+.
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Synthesis of ICG-1. To a solution of 1 (0.2 mg, 0.46 ꢀmol) in DMSO (0.1 mL) was
added N,N-diisopropylethylamine (0.002 mL, 11.5 ꢀmol), followed by ICG-sulfo-OSu
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(0.3 mg, 0.32 ꢀmol). After 2 h at room temperature the reaction mixture was purified by
HPLC as described for 800CW-1 to afford 0.2 mg (60%) of ICG-1 (retention time = 24
min). ESI-Mass calcd for C57H69N5O11S [M]+ 1031.5, found 516.5 [M+H]2+, 1032.0
[M]+.
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Synthesis of Cy7-1. To a solution of 1 (0.5 mg, 1.15 ꢀmol) in DMSO (0.1 mL) was
added N,N-diisopropylethylamine (0.002 mL, 11.5 ꢀmol), followed by the NHS ester of
Cy7 (0.3 mg, 0.37 ꢀmol). After 2 h at room temperature the reaction mixture was
purified by HPLC as described for 800CW-1 to afford 0.2 mg (55%) of Cy7-1 (retention
time = 16 min). ESI-Mass calcd for C47H61N5O14S2 [M]+ 983.4, found 492.5 [M+H]2+,
984.0 [M]+.
Synthesis of Cy5.5-1. To a solution of 1 (0.5 mg, 1.15 ꢀmol) in DMSO (0.1 mL) was
added N,N-diisopropylethylamine (0.002 mL, 11.5 ꢀmol), followed by the NHS ester of
Cy5.5 (0.3 mg, 0.27 ꢀmol). After 2 h at room temperature the reaction mixture was
purified by HPLC as described for 800CW-1 to afford 0.2 mg (62%) of Cy5.5-1
(retention time = 14 min). ESI-Mass calcd for C53H63N5O20S4 [M]+ 1217.3, found 609.4
[M+H]2+, 1217.7 [M]+.
Synthesis of (14S,18S)-1-amino-8,16-dioxo-3,6-dioxa-9,15,17-triazaicosane-14,18,20-
tricarboxylic acid (2). To the tosylate salt of (S)-5-(3-((S)-1,5-bis((4-
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methoxybenzyl)oxy)-1,5-dioxopentan-2-yl)ureido)-6-((4-methoxybenzyl)oxy)-6-
oxohexan-1-aminium 528,29 (0.103 g, 0.121 mmol) in DMF (2 mL) was added Boc-
NH(CH2CH2O)2CH2COOH (0.060 g, 0.135 mmol) and TBTU (0.040g, 0.125 mmol),
followed by N,N-diisopropylethylamine (0.042 mL, 0.241 mmol). After stirring
overnight at room temperature the solvent was evaporated on a rotary evaporator. The
crude material was purified by silica gel column chromatography using
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methanol/methylene chloride (5:95) to give 0.101 g of (14S,18S)-tris(4-methoxybenzyl)
1-amino-8,16-dioxo-3,6-dioxa-9,15,17-triazaicosane-14,18,20-tricarboxylate, 6 in 90%
yield. MALDI-TOF Mass calcd for C47H64N4NaO15 [M+Na]+ 947.4, found 947.1.
Compound 6 was dissolved in a solution of 3% anisole in TFA (1 mL), allowed to react
at room temperature for 10 min, and then concentrated. The crude material was purified
by HPLC [Econosphere C18, 10 ꢀm, 250 x 10 mm, H2O/CH3CN/TFA (92/8/0.1), 4
1
mL/min] to afford 0.035 g (57%) of 2 (retention time = 11 min ). H NMR (400 MHz,
D2O) δ 4.17-4.21 (m, 1H), 4.10-4.13 (m, 1H), 4.00 (s, 2H), 3.67-3.71 (m, 6H), 3.14-3.20
(m, 4H), 2.43-2.46 (m, 2H), 2.08-2.13 (m, 1H), 1.87-1.93 (m, 1H), 1.76-1.79 (m, 1H),
1.63-1.67 (m, 1H), 1.45-1.50 (m, 2H), 1.33-1.40 (m, 2H). 13C NMR (400 MHz, D2O): δ
176.9, 176.8, 176.0, 171.7, 163.0, 162.7, 162.3, 162.0, 158.8, 117.3, 114.4, 69.8, 69.0,
65.9, 52.8, 52.2, 38.6, 38.1, 30.1, 29.6, 27.3, 25.8, 21.5. ESI-Mass calcd for C18H33N4O10
[M]+ 465.2, found 465.2.
Synthesis of 800CW-2. To a solution of compound 2 (0.3 mg, 0.52 ꢀmol) in DMSO
(0.05 mL) was added N,N-diisopropylethylamine (0.002 mL, 11.5 ꢀmol), followed by the
NHS ester of IRDye800CW (0.2 mg, 0.17 ꢀmol). After 2 h at room temperature the
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reaction mixture was purified by HPLC (Econosphere C18, 5ꢀm, 150 × 4.6 mm mobile
phase, A = 50 mM triethylamine acetate buffer (pH 6.0), B = CH3CN; gradient, 0 min =
0% B, 5 min = 0% B, 45 min = 100% B; flow rate, 1 mL/min) to afford 0.2 mg (80%) of
800CW-2 (retention time = 22 min). ESI-Mass calcd for C64H84N6O24S4 [M]+ 1448.4,
found 1448.7.
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Synthesis of 800RS-2. To a solution of 2 (0.3 mg, 0.52 ꢀmol) in DMSO (0.05 mL) was
added N,N-diisopropylethylamine (0.002 mL, 11.4 ꢀmol), followed by the NHS ester of
IRDye800RS (0.2 mg, 0.21 ꢀmol). After 2 h at room temperature the reaction mixture
was purified by HPLC as in 800CW-2 to afford 0.2 mg (75%) of 800RS-2 (retention
time = 28 min). ESI-Mass calcd for C64H84N6O18S2 [M]+ 1288.5, found 1288.9.
Synthesis of ICG-2. To a solution of 2 (0.5 mg, 0.86 ꢀmol) in DMSO (0.1 mL) was
added N,N-diisopropylethylamine (0.002 mL, 11.5 ꢀmol), followed by ICG-sulfo-OSu
(0.3 mg, 0.32 ꢀmol). After 2 h at room temperature the reaction mixture was purified by
HPLC as described for 800CW-1 to afford 0.2 mg (53%) of ICG-2 (retention time = 26
min). ESI-Mass calcd for C63H80N6O14S [M]+ 1176.5, found 589.1 [M+H]2+, 1177.1
[M]+.
Synthesis of Cy7-2. To a solution of 2 (0.5 mg, 0.86 ꢀmol) in DMSO (0.1 mL) was
added N,N-diisopropylethylamine (0.002 mL, 11.5 ꢀmol), followed by the NHS ester of
Cy7 (0.2 mg, 0.24 ꢀmol). After 2 h at room temperature the reaction mixture was
purified by HPLC as in 800CW-1 to afford 0.2 mg (72%) of Cy7-2 (retention time = 17
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min). ESI-Mass calcd for C53H72N6O17S2 [M]+ 1128.4, found 565.0 [M+H]2+, 1129.0
[M]+.
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Synthesis of Cy5.5-2. To a solution of 2 (0.5 mg, 0.86 ꢀmol) in DMSO (0.1 mL) was
added N,N-diisopropylethylamine (0.002 mL, 11.5 ꢀmol), followed by the NHS ester of
Cy5.5 (0.2 mg, 0.18 ꢀmol). After 2 h at room temperature the reaction mixture was
purified by HPLC as in 800CW-1 to afford 0.2 mg (83%) of Cy5.5-2 (retention time = 13
min). ESI-Mass calcd for C59H74N6O23S4 [M]+ 1362.4, found 681.9 [M+H]2+, 1362.7
[M]+.
Synthesis of 800RS-3. To a solution of 39 (0.3 mg, 0.42 ꢀmol) in DMSO (0.1 mL) was
added N,N-diisopropylethylamine (0.002 mL, 11.5 ꢀmol), followed by NHS ester of
IRDye800RS (0.3 mg, 0.31 ꢀmol). After 2 h at room temperature the reaction mixture
was purified by HPLC (column, Econosphere C18 5ꢀ, 150 × 4.6 mm; retention time, 27
min; mobile phase, A = 0.1% TFA in H2O, B = 0.1% TFA in CH3CN; gradient, 0 mins =
0% B, 5 mins = 0% B, 45 mins = 100% B; flow rate, 1 mL/min) to afford 0.3 mg (67%)
of 800RS-3 (retention time = 27 min). ESI-Mass calcd for C72H97N7O19S2 [M]+ 1427.6,
found 714.4 [M+H]2+, 1427.8 [M]+.
Synthesis of ICG-3. To a solution of 3 (0.5 mg, 0.71 ꢀmol) in DMSO (0.1 mL) was
added N,N-diisopropylethylamine (0.002 mL, 11.5 ꢀmol), followed by ICG-Sulfo-OSu
(0.3 mg, 0.32 ꢀmol). After 2 h at room temperature the reaction mixture was purified by
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HPLC as in 800RS-3 to afford 0.3 mg (71%) of ICG-3 (retention time = 32 min). ESI-
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Mass calcd for C71H93N7O15S [M]+ 1315.6, found 1316.0 [M]+.
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Synthesis of Cy7-3. To a solution of 3 (0.5 mg, 0.71 ꢀmol) in DMSO (0.1 mL) was
added N,N-diisopropylethylamine (0.002 mL, 11.5 ꢀmol), followed by the NHS ester of
Cy7 (0.5 mg, 0.61 ꢀmol). After 2 h at room temperature the reaction mixture was
purified by HPLC as in 800RS-3 to afford 0.5 mg (64%) of Cy7-3 (retention time = 19
min). ESI-Mass calcd for C61H85N7O18S2 [M]+ 1267.5, found 634.5 [M+H]2+, 1267.9
[M]+.
Synthesis of Cy5.5-3. To a solution of 3 (0.5 mg, 0.71 ꢀmol) in DMSO (0.1 mL) was
added N,N-diisopropylethylamine (0.002 mL, 11.5 ꢀmol), followed by the NHS ester of
Cy5.5 (0.5 mg, 0.44 ꢀmol). After 2 h at room temperature the reaction mixture was
purified by HPLC as in 800RS-3 to afford 0.6 mg (90%) of Cy5.5-3 (retention time = 18
min). ESI-Mass calcd for C67H87N7O24S4 [M]+ 1501.5 , found 751.4 [M+H]2+, 1501.6
[M]+.
Biology
In Vitro PSMA Inhibition Assay. The PSMA binding affinities of the dye-urea
conjugates for PSMA were measured using the NAALADase assay, as previously
described.30,31 PSMA is known also as glutamate carboxypeptidase II (GCPII), or
NAALADase.32,33 A reaction mixture (total volume of 50 µL) containing [3H]N-acetyl-
aspartylglutamate ([3H]NAAG, 30 nM, 1,850 GBq/mmol) and human recombinant
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GCPII (40 pM final) in Tris-HCl (pH 7.4, 40 mM) containing 1 mM CoCl2 was used.
The reaction was carried out at 37°C for 15 min and stopped with ice-cold sodium
phosphate buffer (pH 7.4, 0.1 M, 50 µL). Blanks were obtained by incubating the
reaction mixture in the presence of 2-phosphonomethyl pentanedioic acid (2-PMPA, 1
uM final), a selective and potent inhibitor of PSMA.32 Dye-urea conjugates and controls
were tested at log-unit final concentrations, that ranged from 100 ꢀM to < fM. A 90 µL
aliquot from each terminated reaction was transferred to a well in a 96-well spin column
containing AG1X8 ion- exchange resin. The plate was centrifuged at 1500 rpm for 5 min
using a Beckman GS-6R centrifuge (Beckman Coulter, Inc., Brea, CA) equipped with a
PTS-2000 rotor. [3H]NAAG bound to the resin and [3H]glutamate eluted in the flow-
through. Columns were then washed twice with formate (1 M, 90 µL) to ensure complete
elution of [3H]glutamate. The flow-through and the washes were collected in a deep 96-
well block. From each well with a total volume of 270 µL, a 200 µL aliquot was
transferred to its respective well in a solid scintillator-coated 96-well plate (Packard,
Meriden, CT) and dried to completion. The radioactivity corresponding to [3H]glutamate
was determined with a scintillation counter (Topcount NXT, Packard, counting efficiency
80%). IC50 curves were generated from CPM results, by use of both Microsoft Office
Excel 2007 and GraphPad Prism 5 programs, with Ki values derived from the IC50
values.34
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Cell Lines and Tumor Models. PSMA+ PC3 PIP and PSMA non-expressing PC3 flu
cell lines were originally a gift from Warren Heston (Cleveland Clinic Foundation35).
Cells were cultured in RPMI 1640 medium (Mediatech, Manassas, VA) containing 10%
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FBS (Sigma) and 1% penicillin-streptomycin (Mediatech) in a humidified incubator
under 5% CO2 at 37◦C. Cells were cultured to 80% confluence and then trypsinized and
collected. Two million cells each of PSMA+ PC3 PIP and PSMA- PC3 flu were re-
suspended in 0.1ml PBS (Mediatech) and injected subcutaneously (s.c) into 4 – 6 week
old male athymic nude mice (NCI, Frederick, MD) in the upper right and left flanks.
Mice were used in imaging studies when tumors reached 3 – 5 mm in diameter.
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In Vivo Imaging and Ex Vivo Biodistribution. In vivo images with compounds Cy5.5-
1, Cy5.5-2 and Cy5.5-3 were acquired on the IVIS Spectrum using an excitation
wavelength of 675 nm and detection of the emission wavelength at 720 nm. The
exposure time for each image acquisition was 1 sec. Images were scaled to the same
maximum intensity using the supplier’s software.
Compounds containing 800CW, 800RS, ICG derivative and Cy7 as fluorophores were
imaged using the Pearl Impulse Imager. The Pearl imager is a dedicated fluorescence
imaging instrument for mice and has fixed excitation wavelengths of 685 nm and 785 nm
and emission wavelengths of 700 nm and 800 nm, respectively, as well as a white-light
overlay. For in vivo studies, stock solutions (~ 1 mM) of dye-urea conjugates were
prepared in H2O (compounds containing 800CW, 800RS, Cy5.5 and Cy7) or DMSO
(compounds containing the ICG derivative) and were diluted with PBS for injection.
After image acquisition at baseline (pre-injection), each mouse was injected
intravenously with 1 nmol of dye-urea conjugate and images were acquired at 5 min, 1 h,
4 h and 24 h time points. Following the 24 h image each mouse was sacrificed by
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cervical dislocation and tumor, muscle, liver, spleen, kidneys and intestine were collected
and assembled on a petri dish for image acquisition. All images were scaled to the same
maximum intensity for direct comparison. For quantification, regions of interest (ROI)
were drawn over the organs displayed in ex vivo images (n = 4) and fluorescence signal
intensity of the organs was calculated using the supplier’s software. Signal intensity of
the muscle was set as background for all calculations. For binding specificity (blocking)
studies, Cy7-3 was co-injected with 1 ꢀmol of (S)-2-(3-((S)-1-carboxy-5-(4-
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iodobenzamido)pentyl)ureido)pentanedioic acid (DCIBzL, a known high-affinity PSMA
inhibitor29) and images were acquired at 1, 4 and 24 h post-injection.
RESULTS AND DISCUSSION
Chemical Synthesis
Compound 1 is urea containing both lysine and glutamate substituents (a lysine-
glutamate urea). It was obtained after deprotection of the tert-butyloxycarbonyl (Boc)
and the p-methoxybenzyl (PMB) groups from compound 428 as outlined in Scheme 1.
Compound 2 has a short polyethylene glycol (PEG) linker. Compound 528 was
conjugated to Boc-NH(CH2CH2O)2CH2COOH to give compound 6, which was then
deprotected to provide compound 2. Compound 3 has a lysine-suberate linker attached to
the lysine-glutamate urea and it was prepared as previously reported.9
The commercially available amine-reactive active esters of IRDye800CW,
IRDye800RS, the indocyanine green (ICG) derivative, Cy7 or Cy5.5 with principal
excitation/emission wavelengths at 774/789, 767/786, 768/804, 743/767 and 675/694 nm,
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respectively, were conjugated with 1, 2 and 3 to produce the dye-PSMA inhibitors shown
in Figure 1. Conjugation of activated esters of the NIR dyes to 1-3 were completed at
room temperature within 2 h. The yields for these conjugates ranged from 53% to 90%.
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In Vitro PSMA Inhibition
The IC50 values of the conjugates were measured using the N-acetylated-α-linked-acidic
dipeptidase (NAALADase) assay30,31 with results presented as Ki values in Table 1. The
Ki values range from 1 – 700 pM, which is similar to other compounds of this
class.9,28,29,36 We observed a trend whereby agents with linkers, such as 800CW-2, ICG-
2, Cy5.5-2, 800CW-3, ICG-3, and Cy5.5-3 had lower Ki values than their counterparts
with no linkers (800CW-1, ICG-1, and Cy5.5-1). However, Cy7 conjugates did not
follow this trend and exhibited similar high affinities independent of the linker. This may
be due to the smaller size of Cy7 compared to Cy5.5 and sulfo-ICG and the greater
flexibility of the polyene moiety in Cy7 compared to the cyclohexene containing polyene
in 800CW and 800RS dyes. Both effects could increase the accessibility of the urea to
the PSMA binding site.
NIR Imaging and Biodistribution
Among the five dyes tested in this study, only the Cy5.5-urea conjugates have excitation
and emission wavelengths below 700 nm. Accordingly, imaging studies with Cy5.5-1,
Cy5.5-2, and Cy5.5-3 were undertaken on the Xenogen IVIS 200 system with excitation
at 675 nm and emission at 720nm. Figure 2 shows images at 24 h after injection of 1
nmol of Cy5.5-1, Cy5.5-2 and Cy5.5-3 in mice with PSMA+ PC3 PIP and PSMA- PC3
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flu tumors. All three compounds demonstrated high uptake within PSMA+ PC3 PIP
tumors and little uptake in PSMA- PC3 flu tumors. The relative degree of observed
PSMA+ PC3 PIP tumor uptake was in the order: Cy5.5-3 > Cy5.5-2 > Cy5.5-1. Renal
uptake is due to high expression of PSMA within proximal renal tubules as well as to
excretion, as previously shown.37-39 Imaging of compounds containing 800CW, 800RS,
the ICG derivative and Cy7 as fluorophores were conducted using the Pearl Imager,
which has a set excitation wavelength at 785 nm and emission at 800 nm. These dye-
urea conjugates have excitation and emission wavelengths above 700 nm, which allow
for better depth of light penetration than compounds that emit at lower wavelengths and
minimal autofluorescence. Figure 3 shows typical whole body and excised organ
imaging of mice with PSMA+ PC3 PIP and PSMA- PC3 flu tumors at 24 h after injection
of 1 nmol of the 800CW, 800RS, the ICG derivative or Cy7-urea conjugates. All
compounds demonstrated PSMA+ PC3 PIP tumor uptake with little PSMA- PC3 flu
tumor uptake, indicating target selectivity in vivo.
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These images also demonstrate that the choice of dye and linking group have significant
effects on biodistribution. Intense fluorescence signals were observed in both tumor and
kidneys for all three IRDye800CW conjugates (800CW-1, 800CW-2 and 800CW-3) with
800CW-3 having much lower normal tissue uptake than the other two compounds.
IRDye800RS has a similar structure, but with two less sulfonate groups compared to
IRDye800CW. PSMA+ PIP tumor uptake was observed in all three 800RS-urea
conjugates. However, the fluorescence intensities were significantly lower than for the
corresponding 800CW conjugates. Among those three 800RS-urea conjugates, 800RS-3,
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which has a lysine suberate linker, showed highest PSMA+ PIP tumor uptake. Similarly,
among the three ICG-urea conjugates, ICG-3, gave the highest PSMA+ PIP tumor
uptake. ICG-3 also demonstrated higher PSMA+ PIP tumor uptake compared to kidney,
which differs from 800CW-3 and 800RS-3, which had roughly equivalent tumor and
kidney uptake. Cy7-2 and Cy7-3 had significantly higher PSMA+ PIP tumor uptake than
uptake in kidneys. They also demonstrated the lowest normal tissue uptake comparing to
the other compounds with the same linker. From this preliminary evaluation we chose
800CW and Cy7 conjugates for further study, where the organs of 4 mice/agent were
harvested and imaged at 24 h post-administration of imaging agent. Fluorescence
intensity per organ is shown in Figure 4. In both the Cy7 series and the IRDye800CW
series the degree of PSMA+ PIP tumor uptake was 800CW-3 or Cy7-3 ≈ 800CW-2 or
Cy7-2 > 800CW-1 or Cy7-1, confirming the importance of the linker moiety for
modifying pharmacokinetics. Tumor to kidney ratios for Cy7-2 and Cy7-3 were higher
than for 800CW-2 and 800CW-3. The fluorescence intensities of all organs except the
gastrointestinal tract for Cy7 conjugates were lower than for the corresponding 800CW
conjugates. This is most likely because the Pearl Imager has a fixed excitation maximum
at 785 nm and collects emission at 820 nm, which is further from the excitation and
emission maxima of Cy7 (743/767) than those for IRDye800CW (774/789).
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In order to demonstrate the PSMA-binding specificity of the PSMA inhibitor-dye
conjugates, DCIBzL was co-administered with Cy7-3. As shown in Figure 5, all
emission from the PSMA+ target tumor was blocked with concurrent DCIBzL
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administration, indicating specific binding. The excised organ imaging results are
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The active site of PSMA possesses two binding sites, comprised of a pharmacophore
(S1’) site and an amphiphilic, non-pharmacophore (S1) site.40 The S1’ site is generated
by amino acid residues highly sensitive to structural modification of potential ligands,
demonstrating a strong preference for glutamate or glutamate-like residues. Compared to
the S1’ site, the S1 site is more promiscuous. A tunnel-like region (~ 20 Å) linked to the
S1 site is projected toward the surface of the enzyme. Utilizing the tunnel region we
have attached bulky chelated metals to the urea-based specificity-conferring moiety
through a long linker to maintain the chelated metal entirely outside of protein. In so
doing we have produced a small array of PSMA-targeted radiotracers capable of imaging
PSMA in experimental models of prostate cancer.28,36,41 Similarly, by attaching bulky
fluorophores to the urea inhibitors through a linker, such as in dye-2 or dye-3 conjugates,
the fluorophores can be situated outside of the active site, allowing maximum interaction
with the urea-based specificity conferring portion of the agent. This effect is most
noticeable in the higher tumor uptake demonstrated by 800RS-2, 800RS-3, ICG-2, ICG-
3, and Cy5.5-3 compared to dye-1conjugates 800RS-1, ICG-1, and Cy5.5-1.
Recently Nakajima et al. have used the anti-PSMA antibody J591 conjugated to ICG to
visualize PSMA+ tumors in whole mice at up to 10 days after administration.10 We and
others have focused on developing low molecular weight optical imaging agents for
PSMA, in part to avoid the longer biological half-lives of antibody-based methods, to
enable imaging at shorter times after administration.9,23,24,42-44 However, to date most
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such low molecular weight agents have only been used in vitro. Humblet et al. has
reported in vivo imaging of PSMA-binding NIR-emitting phosphonate derivatives,
however at 20 sec, the time of maximum PSMA-positive tumor uptake, the tumor to
background ratio is less than two.24 Previously we were able to image PSMA+ tissues in
experimental models of prostate cancer using the targeted, IRDye800CW-based agent,
YC-27 (800CW-3).9 Here we used the same urea-based targeting moiety but expanded to
the small series reported, investigating the pharmacokinetic effects of different linkers
and fluorophores.
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CONCLUSION
Compounds 2 and 3, which contain PEG and lysine-suberate linkers, respectively,
provide useful scaffolds for optical imaging of PSMA. Although IRDye800CW and Cy7
conjugates 800CW-2, 800CW-3, Cy7-2, and Cy7-3 demonstrated high, PSMA-specific
uptake in vivo at 24 h post-injection, the exact choice of dye for intra-operative imaging
will depend upon the specifications of the camera used for detection in the operating
suite. Nevertheless, this small series demonstrates that a variety of dyes with different
absorption and emission spectra can be accommodated.
ACKNOWLEDGMENTS
We thank CA134675 and the AdMeTech Foundation for financial support and Dr. Ron
Rodriguez for helpful discussions.
SUPPORTING INFORMATION
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HPLC conditions and traces for the final compounds, ligated to the commercial and
previously reported NIR dyes used herein, are provided: 800CW-1; 800CW-2; 800CW-3;
800RS-1; 800RS-2; 800RS-3; ICG-1; ICG-2; ICG-3; Cy7-1; Cy7-2; Cy7-3; Cy5.5-1;
Cy5.5-2; Cy5.5-3. This material is available free of charge via the Internet at
http://pubs.acs.org.
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ABBREVIATIONS
NIR: near-infrared
ICG: indocyanine green
PSMA: prostate-specific membrane antigen
NAAG: N-acetylaspartyl glutamate
NAALADase: N-acetylated-α-linked-acidic dipeptidase
Boc: tert-butyloxycarbonyl
PMB: p-methoxybenzyl
PEG: polyethylene glycol
NHS: N-hydroxysuccinimide
DCIBzL: (S)-2-(3-((S)-1-carboxy-5-(4-iodobenzamido)pentyl)ureido)pentanedioic acid
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FIGURES
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O
O
H
N
H
N
H
N
R
O
NH
O
NH
R
R
NH
O
H
OH
O
H
OH
OH
HOOC
O
O
H
OH
OH
O
O
H
O
HO
OH
HO
HO
N
H
N
H
N
H
N
H
N
H
N
H
H
H
H
O
O
O
O
9
O
O
1: R = H
Dye-1: R = Dye
3: R = H
3
: R = Dye
2
Dye-2: R
: R =
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Dye-
= Dye
-
SO3
HO3
S
-
HO3
S
HO3
S
SO3
O
O
N
N+
N+
N
N+
Dye:
N
-O3
S
HO3
S
O
O
IRDye800RS
IRDye800CW
ICG derivative
O
SO3
H
HO3
S
SO3
H
-O3
S
SO3
H
-O3
S
N
N+
N+
N
O
Cy7
Cy5.5
O
Figure 1. Structures of PSMA-based near-infrared fluorescent imaging agents.
Figure 2. Whole body and ex vivo organ imaging of mice with PSMA+ PC3 PIP and
PSMA- PC3 flu tumors at 24 h post-injection of 1 nmol of (a) Cy5.5-1, (b) Cy5.5-2 and
(c) Cy5.5-3.
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Figure 3. Whole body and ex vivo organ imaging of mice with PSMA+ PC3 PIP and
PSMA- PC3 flu tumors at 24 h post-injection of 1 nmol of the indicated dye-urea
conjugates.
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