68Ga-Labeled Inhibitors of Prostate-Specific Membrane antigen (PSMA) for Imaging Prostate Cancer

doi:10.1021/jm100623e

J Med Chem. Author manuscript; available in PMC 2012 May 2.

Published in final edited form as:

J Med Chem. 2010 July 22; 53(14): 5333–5341.

doi: 10.1021/jm100623e

PMCID: PMC3341619

NIHMSID: NIHMS365978

⁶⁸Ga-Labeled Inhibitors of Prostate-Specific Membrane antigen (PSMA) for Imaging Prostate Cancer

Sangeeta Ray Banerjee, Mrudula Pullambhatla, Youngjoo Byun, Sridhar Nimmagadda, Gilbert Green, James J. Fox, Andrew Horti, Ronnie C. Mease, and Martin G. Pomper

Russell H. Morgan Department of Radiology and Radiological Sciences, Johns Hopkins Medical Institutions, Baltimore, MD 21231

Corresponding Author: Martin G. Pomper, M.D., Ph.D., Johns Hopkins Medical Institutions, 1550 Orleans Street, 492 CRB II, Baltimore, MD 21231, 410-955-2789 (T), 443-817-0990 (F), Email: mpomper/at/jhmi.edu

The publisher's final edited version of this article is available at J Med Chem

See other articles in PMC that cite the published article.

Abstract

Gallium-68 is a generator-produced radionuclide for positron emission tomography (PET) that is being increasingly used for radiolabeling of tumor-targeting peptides. Compounds [⁶⁸Ga]3 and [⁶⁸Ga]6 are high-affinity, urea-based inhibitors of the prostate-specific membrane antigen (PSMA) that were synthesized in decay-uncorrected yields ranging from 60 – 70% and radiochemical purities of more than 99%. Compound [⁶⁸Ga]3 demonstrated 3.78 ± 0.90 percent injected dose per gram of tissue (%ID/g) within PSMA+ PIP tumor at 30 min post-injection, while [⁶⁸Ga]6 showed a two hour PSMA+ PIP tumor uptake value of 3.29 ± 0.77%ID/g. Target (PSMA+ PIP) to non-target (PSMA− flu) ratios were 4.6 and 18.3, respectively, at those time points. Both compounds delineated tumor clearly by small animal PET. The urea series of imaging agents for PSMA can be radiolabeled with ⁶⁸Ga, a cyclotron-free isotope useful for clinical PET studies, with maintenance of target specificity.

Keywords: gallium, molecular imaging, positron emission tomography, prostate-specific membrane antigen, radiopharmaceutical

Introduction

Prostate cancer is the most commonly diagnosed malignancy and the second leading cause of cancer-related death in men in the United States.¹ In 2009, approximately 192,000 men were diagnosed with prostate cancer with 27,000 succumbing to the disease. The integral membrane protein prostate-specific membrane antigen (PSMA) is becoming increasingly recognized as a viable target for imaging and therapy of prostate and other forms of cancer.^{2, 3}

Because of its similarity to Fe(III), Ga(III) complexes are emerging as an interesting alternative to Pt-based anticancer agents.^4–6 From a diagnostic standpoint, positron-emitting versions of Ga(III) can be used for tumor imaging.^7–9 Recently, the application of ⁶⁸Ga-labeled peptides has attracted considerable interest for cancer imaging because of the physical characteristics of ⁶⁸Ga.¹⁰ ⁶⁸Ga is available from an in-house ⁶⁸Ge/⁶⁸Ga generator (⁶⁸Ge, t_1/2 = 270.8 day), which renders it independent of an onsite cyclotron. Therefore, ⁶⁸Ga-based PET agents possess significant commercial potential and serve as a convenient alternative to cyclotron-based isotopes for positron emission tomography (PET), such as ¹⁸F or ¹²⁴I. ⁶⁸Ga has a high positron-emitting fraction (89% of its total decay). The maximum positron energy of ⁶⁸Ga (max. energy = 1.92 MeV, mean = 0.89 MeV) is higher than that of ¹⁸F (max = 0.63 MeV, mean = 0.25 MeV). However, a study of spatial resolution using Monte Carlo analysis revealed that under the assumption of 3 mm spatial resolution for most PET detectors, the full-width-at-half-maximum (FWHM) of ¹⁸F and ⁶⁸Ga are indistinguishable in soft tissue (3.01 mm vs. 3.09 mm).⁹ That finding implies that with the standard spatial resolution of 5 to 7 mm for current clinical scanners, image quality using ⁶⁸Ga-based radiotracers will likely be indistinguishable from that of ¹⁸F-based agents, stimulating interest in the development of ⁶⁸Ga-labeled compounds for medical imaging.^7–9 With a physical half-life of 68 min, ⁶⁸Ga is also matched nicely to the pharmacokinetics of many peptides used for imaging. Furthermore, ⁶⁸Ga is introduced to biomolecules through macrocyclic chelators, which allows possible kit formulation and wide availability of the corresponding imaging agents.

We and others have previously demonstrated the ability to image PSMA-expressing prostate tumor xenografts with radiohalogenated, urea-based, low molecular weight inhibitors of PSMA.^{11, 12, 13, 14, 15} Recently, we have extended that work to include the radiometal ^99mTc via a coordinated, ^99mTc tricarbonyl moiety.¹⁶ To retain the binding affinity of those inhibitors to PSMA a linker moiety was introduced between the amino functionalized PSMA urea and the metal chelator (Figure 1). A similar approach by Kularatne et al. produced ^99mTc-oxo labeled inhibitors.^{14, 15} We have now extended this work further to include ⁶⁸Ga for PET imaging. Gallium(III) ion forms a stable complex (formation constant, logK_ML = 21.33) with the commercially available, widely used multidentate chelating agent, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA).¹⁷ This report describes the synthesis and in vitro binding of two new ⁶⁸Ga-labeled, conjugated PSMA inhibitors, [⁶⁸Ga]3 and [⁶⁸Ga]6 (Figure 1), as well the biodistribution and in vivo imaging studies of these compounds. The chelating agent we have employed is the triacetic acid mono-amide of DOTA. Few ⁶⁸Ga-labeled, mechanism-based radiotracers for prostate cancer have been reported previously, and none for PSMA or that approach such low molecular weights as these.

Figure 1

Urea-based PSMA radioligands ^99mTcL1, [⁶⁸Ga]3 and [⁶⁸Ga]6.

Results

Chemical and Radiochemical Syntheses

DOTA-conjugated urea inhibitors

Key N-hydroxysuccinimide (NHS) ester intermediate 1, (Scheme 1) was prepared following our previous report.¹⁶ Compound 1 was then conjugated with the α-amine of H-Lys(Boc)-OBz¹⁸ followed by simultaneous removal of Boc and PMB (p-methoxybenzyl) groups using a solution of trifluoracetic acid (TFA)/CH₂Cl₂ at 25°C to produce 2. The primary amine of 2 was then conjugated with DOTA-NHS (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono(N-hydroxysuccinimide ester)) to obtain 3, in ~ 40% yield after purification by high-performance liquid chromatography (HPLC). NMR and mass spectrometry (MS) were used to confirm the identity of 3.

Scheme 1

Synthesis of DOTA conjugated PSMA inhibitor 6 was performed by using standard fluorenylmethoxycarbonyl (Fmoc) solid phase peptide synthesis (SPPS) starting from Fmoc-Lys(Boc)-Wang resin according to Scheme 2. After conjugating two phenylalanine residues with the resin bound lysine, DOTA was conjugated at the N-terminal of the second phenylalanine residue after which the compound was cleaved from the resin by a 1:1 mixture of TFA:CH₂Cl₂ to produce 4. The free ε-amine of lysine was then conjugated with 5,¹⁹ which was prepared from 1 by PMB cleavage, to produce 6. Compound 6 was characterized through standard spectroscopic techniques.

Scheme 2

A stable gallium isotope (^69,71Ga) was introduced to the urea-DOTA conjugate by incubation of 3 or 6 with an aqueous solution of GaCl₃ at 95°C for 10 min. Compounds [^69,71Ga]3 and [^69,71Ga]6, were characterized by standard spectroscopic analyses. The proposed structures of compounds [^69,71Ga]3 and [^69,71Ga]6, as shown in Schemes 1 and and22 and Figure 1, were based on the reported X-ray crystal structures of gallium-DOTA compounds described by Maecke et al^{20, 21} and Doyle et al.²² The mass spectra of the Ga compounds showed the expected isotope distribution pattern for natural gallium, which is a mixture ⁶⁹Ga (60.11%) and ⁷¹Ga (39.89%).²³ The stable gallium-labeled conjugates were used as authentic reference material for the chromatographic analysis of the radiolabeling reactions to identify the corresponding ⁶⁸Ga-labeled compounds.

Radiochemistry

The ⁶⁸Ga(III) was eluted from the ⁶⁸Ge/⁶⁸Ga generator using ~ 6 – 7 mL of a solution of 0.1 N hydrochloric acid (HCl). To achieve high specificity radioactivity, radioactive material was preconcentrated and purified on a cation exchange resin following a literature method.²⁴ The ⁶⁸Ga(III) was eluted from the resin with 400 µL of an 97.6% acetone/0.05 N HCl mixture (pH, 2.30 ± 0.05) and was used immediately for aqueous radiolabeling of 3 and 6. No buffer solution was added. The radiolabeling was performed at 90 – 95°C for 10 min with decay-uncorrected yields ranging from 60 – 70% and radiochemical purities of more than 99%. On analysis of the reaction mixture by HPLC, the retention time of the radiolabeled compound was slightly longer than the corresponding free ligand. The specific radioactivity of purified [⁶⁸Ga]3 and [⁶⁸Ga]6 was between 3.0 and 6.0 MBq/nmol. The log P_{octanol/water} values for [⁶⁸Ga]3 and [⁶⁸Ga]6 were approximately −3.9 as determined by the shake-flask method.²⁵ However, using an HPLC method, we found that the HPLC retention times for 6 (28 min) and [^69/71Ga]6 (32 min) were longer than for 3 (19 min) and [^69/71Ga]3 (24 min). It is evident that 6 and the corresponding gallium compound were more lipophilic than 3 and its gallium–labeled analog, which is reasonable in light of the presence of two phenylalanine residues in the long linker of 6, while 3 has only one lysine residue protected as the benzyl ester. Interestingly, our previous lead compound, ^99mTcL1,¹⁶ was found to be much more lipophilic than either of these gallium compounds, with log P_{octanol/water}~ −3.1, possibly because of its organometallic tricarbonyl core as well as the presence of the lipophilic bispyridyl chelating agent.

Biology

Cell binding assay

K_i values for 3, [^{69, 71}Ga]3, 6 and [^{69, 71}Ga]6 were determined using a fluorescence-based PSMA inhibition assay.²⁶ All compounds were found to be potent inhibitors of PSMA, as we reported earlier for ^99mTcL1 and related compounds.¹⁶ Compounds 3 and [^69,71Ga]3 had inhibitory capacities of 2.9 nM and 29 nM, respectively. For 6 and [^{69, 71}Ga]6, values were 1.23 nM and 0.44 nM, respectively.

Biodistribution

Compound [⁶⁸Ga]3 was assessed for its pharmacokinetics ex vivo in severe-combined immunodeficient (SCID) mice bearing both PSMA+ PC3-PIP and PSMA− PC3-flu xenografts.²⁷ Table 1 shows the percent injected dose per gram (%ID/g) of radiotracer in selected organs for [⁶⁸Ga]3. Compound [⁶⁸Ga]3 showed clear PSMA-dependent binding in PSMA+ PC3 PIP xenografts, reaching a maximum uptake of 3.78 ± 0.90 (SEM) %ID/g at 30 min post-injection (pi). The blood, spleen and kidney displayed highest uptake at 30 min. By 60 min, the urinary bladder showed highest uptake, however, this uptake represents excretion at all time points. The high values noted in kidney are partially due to high expression of PSMA within proximal renal tubules.^{28, 29} Rapid clearance from the kidneys was demonstrated, decreasing from 97.19 ± 16.07 %ID/g at 30 min to 2.31 ± 0.11%ID/g at 3 h. The radioactivity in the PSMA+ PIP tumor cleared more slowly, from its aforementioned value at 30 min to 1.08 ± 0.19 %ID/g at 3 h.

Table 1

Ex Vivo Tissue Biodistribution of [⁶⁸Ga]3

Compound [⁶⁸Ga]6 was also investigated for its pharmacokinetic characteristics in tumor-bearing mice at 5 min, 1 h, 2 h and 3 h pi. Table 2 shows the %ID/g of radiotracer in selected organs for [⁶⁸Ga]6. As for [⁶⁸Ga]3, [⁶⁸Ga]6 showed PSMA-dependent tumor uptake. After a peak, flow-related, uptake at 5 min pi of 6.61 ± 0.55%, [⁶⁸Ga]6 demonstrated a 2 h tumor uptake value of 3.29 ± 0.77%, which dropped to 1.80 ± 0.16% at 3 h. Uptake in blood was high at 5 min and rapidly washed out within 1 h. Non-target organs such as kidney, spleen and lung showed high uptake at 5 min and rapidly washed out with time. With the exception of the kidneys and spleen, clearance from blood and normal organs was faster for [⁶⁸Ga]6 than for [⁶⁸Ga]3. Again, high kidney uptake is associated with high expression of PSMA within proximal renal tubules.^{28, 29} Similar to [⁶⁸Ga]3, [⁶⁸Ga]6 demonstrated faster clearance of radioactivity from kidney than from the PSMA+ tumor. However, the rate of clearance from kidney for [⁶⁸Ga]6 was much slower than for [⁶⁸Ga]3, i.e., 65 ± 12% at 5 min pi and 10 ± 1.22% at 3 h.

Table 2

Ex Vivo Tissue Biodistribution of [⁶⁸Ga]6

Small animal PET imaging

Intense radiotracer uptake was seen only in the kidneys and tumor for both [⁶⁸Ga]3 (Figure 2) and [⁶⁸Ga]6 (Figure 3). As noted above for the ex vivo study, the intense renal uptake was partially due to specific binding of the radiotracer to proximal renal tubules^{28, 29} as well as to excretion of this hydrophilic compound. Apart from the kidneys, only the PSMA+ tumor demonstrated significant radiotracer uptake.

Figure 2

GE eXplore VISTA pseudodynamic PET image (co-registered with the corresponding CT image) of a PSMA+ LNCaP tumor-bearing mouse injected intravenously with 0.2 mCi (7.4 MBq) of [⁶⁸Ga]3.

Figure 3

GE eXplore VISTA PET image (co-registered with the corresponding CT image) of a PSMA+ PIP and PSMA− flu tumor-bearing mouse injected intravenously with 0.2 mCi (7.4 MBq) of [⁶⁸Ga]6.

Discussion

Because of its demonstrated clinical utility and the appearance of dual modality [PET/computed tomography (CT)] systems, clinical PET imaging has been accelerating worldwide and may soon become the dominant technique in nuclear medicine. PET isotopes tend to be short-lived and enable synthesis of “physiologic” radiotracers, namely, those that incorporate ¹⁵O, ¹³N or ¹¹C, enabling precise conformity to the tracer principle. Being essentially isosteric to H, ¹⁸F enables nearly tracer-level studies, with important caveats, particularly for [¹⁸F]fluorodeoxyglucose (FDG), which is by far the most commonly used radiopharmaceutical for PET. But, in part because FDG does not accumulate well within many tumor types,³⁰ including prostate cancer,^{30, 31} there has been a sustained effort in the development of radiometallated peptides, often employing ^99mTc, that target G-protein coupled receptors. Gallium-68 provides a link between PET and single photon emission computed tomography (SPECT) since metal chelating methodology needed for ^99mTc can also be applied to ⁶⁸Ga. A further analogy is the convenience of a ⁶⁸Ge/⁶⁸Ga generator (PET), as with ⁹⁹Mo/^99mTc (SPECT), to provide readily available isotope, with no need for an in-house cyclotron. Although ¹⁸F-labeled, low molecular weight PSMA inhibitors have shown promise in preclinical imaging studies,^{13, 32} the ready availability of generator-produced ⁶⁸Ga and the logical extension to PET from our published ^99mTc-labeled series of PSMA-binding radiometallated imaging agents¹⁶ provide the rationale for this study.

As for the ^99mTc-labeled agents, the strategy we employed involves a tripartite imaging agent containing a PSMA targeting moiety, a linker and a chelator for ⁶⁸Ga. The linker is necessary to enable productive binding by directing the ⁶⁸Ga-chelate complex through a 20Å tunnel away from the active site. Because of its ability to chelate metals with a +3 oxidation state and its clinical track record we used DOTA as the chelator for both [⁶⁸Ga]3 and [⁶⁸Ga]6.^33–39 Both are also derived from a lysine-urea-glutamate construct that confers PSMA specificity. That still leaves significant structural differences, which are confined to the linker, between those two compounds. Those differences include two phenylalanines for [⁶⁸Ga]6 relative to [⁶⁸Ga]3, while the latter compound possesses one benzyl-protected lysine within the linker. Because of the strict structural requirements of the S1’ (pharmacophore) pocket in which the glutamate moiety resides,⁴⁰ and the need for at least one additional carboxylate (derived from lysine), modification of the linker is the best option to enable pharmacokinetic optimization of this series. A careful balance is sought whereby sufficient localization of the radiotracer to the tumor is needed, favoring higher hydrophobicity, while washout from non-target sites such as liver and intestine is also desired, favoring higher hydrophilicity. The benzyl group was initially added to [⁶⁸Ga]3 to provide a chromophore to facilitate purification, but the phenylalanines in [⁶⁸Ga]6 were added to offset the high hydrophilicity of these compounds, potentially enabling longer and/or higher tumor sequestration, as originally proposed in a previous report.¹⁴ The need for long retention times, while not necessary when using ⁶⁸Ga (physical half-life = 68 min), may be needed for longer lived isotopes, such as ¹¹¹In, or for therapeutic radiometals. However, even addition of two phenylalanines was not able to provide a compound as lipophilic as our previously published SPECT agent, ^99mTcL1.

Figures 2 and and33 demonstrate the high target selectivity of [⁶⁸Ga]3 and [⁶⁸Ga]6 by delineating the PSMA+ tumors as well as kidney, which is a PSMA+ structure. Because we anticipate that most metastatic foci, for which these compounds are designed, will be in bone or lymph nodes (particularly those in pelvis), we do not anticipate that renal uptake will provide a significant confound for these agents. Although a PSMA− control tumor was not included in Figure 2, a separate blocking study was performed for [⁶⁸Ga]3, in which an animal pre-treated with 50 mg/kg of the known PSMA-binding ligand, 2-(phosphonomethyl)pentanedioic acid (2-PMPA),⁴¹ did not demonstrate PSMA+ tumor uptake (see Supplementary Information Available), attesting to the binding specificity of this compound. The more quantitative, ex vivo studies of [⁶⁸Ga]3 and [⁶⁸Ga]6 further support high PSMA target specificity, demonstrating target-to-nontarget (PIP/flu) ratios of approximately 5 and 18 at 1 h and 2 h pi, respectively. One hour and 2 h PSMA+ tumor uptake values for these compounds, 3.32 ± 0.33% and 3.29 ± 0.77%, respectively, for [⁶⁸Ga]3 and [⁶⁸Ga]6, are comparable to other radiometallated PSMA inhibitors we have developed.¹⁶ As shown in Figures 2 and and33 those values are sufficient for clear tumor imaging. Notably, PIP tumors contain about one order of magnitude lower PSMA than LNCaP tumors (data not shown), which are often employed to assess for binding specificity of PSMA-targeting agents. We generally prefer the PIP/flu comparison as both are derived from PC-3 cells, providing a more controlled study.

Conclusions

Compounds [⁶⁸Ga]3 and [⁶⁸Ga]6 demonstrate PSMA-specific tumor imaging in vivo. Because of higher target-to-nontarget ratios with comparable absolute uptake values to [⁶⁸Ga]3, [⁶⁸Ga]6 will be pursued in additional animal models and for toxicity testing en route to clinical translation. In this manner we hope to add this cyclotron-independent radiopharmaceutical to the array of emerging agents for imaging prostate cancer.

Experimental Section

General Procedures

Solvents and chemicals obtained from commercial sources were of analytical grade or better and used without further purification. All experiments were performed in duplicate or triplicate to ensure reproducibility. Analytical thin-layer chromatography (TLC) was performed using Aldrich aluminum-backed 0.2 mm silica gel Z19, 329-1 plates and visualized by ultraviolet light (254 nm), I₂ and 1% ninhydrin in EtOH. Flash chromatography was performed using silica gel purchased from Bodman (Aston PA), MP SiliTech 32–63 D 60Å. ¹H NMR spectra were recorded on a Bruker ultrashield™ 400 MHz spectrometer. Chemical shifts (δ) are reported in ppm downfield by reference to proton resonances resulting from incomplete deuteration of the NMR solvent. Low resolution ESI mass spectra were obtained on a Bruker Daltonics Esquire 3000 Plus spectrometer. High resolution mass spectra were obtained at the University of Notre Dame Mass Spectrometry & Proteomics Facility, Notre Dame, IN using ESI either by direct infusion on a Bruker micrOTOF-II or by LC elution via an ultra-high pressure Dionex RSLC with a C18 column coupled with a Bruker micrOTOF-Q II. High-performance liquid chromatographic purification of new compounds, 3, [^69/71Ga]3, 6, [^69/71Ga]6 and [⁶⁸Ga]3, was performed using a Phenomenex C₁₈ Luna 10 × 250 mm² column on a Waters 600E Delta LC system with a Waters 486 tunable absorbance UV/Vis detector, both controlled by Empower software. For purification of radiolabeled [⁶⁸Ga]6, a Varian Microsorb-Mv C₁₈ 250 × 4.6 mm² column was used. HPLC was performed using the following isocratic conditions: For Method 1, the mobile phase was 80% solvent A (0.1% TFA in water) and 20% solvent B (0.1% TFA in CH₃CN), flow rate 4 mL/min; for Method 2, the mobile phase was 80% solvent A and 20% solvent B, flow rate 1 mL/min. Method 1 was used for purification of compounds 3, [^69/71Ga]3, 6, [^69/71Ga]6 and [⁶⁸Ga]3. For purification of [⁶⁸Ga]6, Method 2 was used. For radiosynthetic purification, HPLC was performed on a Varian Prostar System (Palo Alto, CA), equipped with a model 490 UV absorbance detector and a Bioscan NaI scintillation detector connected to a Bioscan Flow-count system. All final compounds were obtained in > 95% purity, as determined by HPLC.

2-{3-[5-(7-{1-Benzyloxycarbonyl-5-[2-(4,7,10-tris-carboxymethyl-1,4,7,10tetraazacyclododec-1-yl)-acetylamino]-pentylcarbamoyl}-heptanoylamino)-1-carboxy-pentyl]-ureido}-pentanedioic acid, 3

Compound 3 was prepared in three steps according to Scheme 1. Compound 1 was prepared according to a literature method.¹⁶ To a solution of 1 (100 mg, 0.11 mmol in 5 mL DMF) was added H-Lys(Boc)-OBz (36 mg, 0.11 mmol).¹⁸ The solution was stirred for 16 h at ambient temperature. The solvent was removed under vacuum. The solid residue thus obtained was dissolved in 10 mL ethyl acetate and extracted with 3 × 10 mL water. The organic layer was dried under vacuum to provide a colorless solid ESIMS: 1154 [M+1]⁺. This crude compound was dissolved in 3 mL CHCl₃ followed by addition of 3 mL TFA at 0°C. The solution was allowed to stir overnight at ambient temperature. The volume of the solution was reduced under vacuum and the solid residue was washed with 3 × 5 mL CH₂Cl₂ to remove impurities. The colorless solid residue, 2, was dried under vacuum. The crude yield for 2 was 80 mg. Compound 2 was purified further by using a 2 g Sep Pak C18 cartridge with a solution of 85/15 water/acetonitrile (0.1% TFA in each). ¹H NMR (D₂O, δ): 7.5 (bm, 5H, Ph), 5.12 (s, 2H, PhCH₂), 4.27 (m, 1H, HC(NH)CO₂(Glu)), 4.16(m, 1H, HC(NH)CO₂(Lys)), 3.99 (m, 1H, HC(NH)CO₂(Lys-linker)), 3.08 (m, 4H, H₂CNH(Lys), H₂CNH(Lys-linker)), 2.39(t, 2H, H₂CCO-linker), 2.21 (m, 2H, H₂CCO₂(Glu)), 2.19(t, 2H, H₂CCO-linker), 1.89-1.57(m, 6H, H₂CCH(Glu), H₂CCH(Lys), H₂CCH(Lys-linker)), 1.43-1.16 (m, 16H, (CH₂)₂(Lys), (CH₂)₂(Lys-linker), (CH₂)₄ (linker)). ESIMS: 694 [M+1]⁺.

To a solution of DOTA-mono-NHS (54 mg, 0.11 mmol in 5 mL DMF) was added 2 (80mg, 0.08 mmol) and TEA (60 µL, 0.43 mmol) and the solution was allowed to stir for 16 h at ambient temperature. Solvent was removed under vacuum and the crude solid, 3, was purified by HPLC Method 1, retention time 19 min. Yield: ~ 40%. ¹H NMR (D₂O) δ: 7.88 (m, 5H, Ph), 5.10 (s, 2H, H₂CPh), 4.26 (m, 1H, HC(NH)CO₂(Glu)), 4.16(m, 1H, HC(NH)CO₂(Lys)), 4.06 (m, 1H, HC(NH)CO₂(Lys-linker), 3.66 (m, 8H, H₂CCO₂), 3.18 (m, 20H, N(CH₂)₂N(DOTA), H₂CNH(Lys), H₂CNH(Lys-linker)), 2.39(t, 2H, H₂CCO-linker), 2.15 (m, 2H, H₂CCO₂(Glu)), 2.07(t, 2H, H₂CCONH-linker),1.85-1.55(m, 6H, H₂CCH(Glu), H₂CCH(Lys), H₂CCH(Lys-linker)), 1.41-1.14 (m, 16H, (CH₂)₂(Lys), (CH₂)₂(Lys-linker), (CH₂)₄ (linker). ¹³C (D₂O) δ: 177.8 (CO₂H), 177.6 (CO₂H), 177.5 (CO₂H), 177.1 (CO₂H), 176.3 (CO₂H), 174.2(CO₂CH₂Ph), 173.9 (CONH), 159.8,(NHCONH), 135.5 (C, Ph), 128.9(CH, Ph), 128.5 (CH, Ph),128.1(CH, Ph), 67.3 (CH₂Ph), 55.5 (CH₂CO₂H), 53.4 (CH, Glu), 53.2, 53.1(CH, Lys, Lys-linker), 52.5, 52.3 (CH₂, DOTA), 39.0 (CH₂NH, Lys), 38.9 (CH₂NH, Lys-linker), 35.5 (CH₂CO, linker), 35.4 (CH₂CO, linker), 30.7(CH₂CO, (Glu)), 28.0 (CH₂CH (Glu)), 27.4, 27.3, 27.1, 26.4, 25.1 (CH₂ (linker), (Lys), (Lys-linker)), 22.3, 22.2(CH₂(Lys), CH₂(Lys-linker)). ESIMS: 1080[M+1]⁺, HRESI⁺-MS: Calcd. For C₄₉H₇₇N₉O₁₈, 1080.5487 [M+H], found: 1080.5459.

2-{3-[5-(7-{1-Benzyloxycarbonyl-5-[2-(4,7,10-tris-carboxymethyl-1,4,7,10tetraazacyclododec-1-yl)-acetylamino]-pentylcarbamoyl}-heptanoylamino)-1-carboxy-pentyl]-ureido}-pentanedioic acid Gallium (III), [^69/71Ga]3

To a solution of GaNO₃ (5 mg, 20 µmol) in deionized water was added compound 3 (20 mg, 20 µmol) in 1 mL deionized water. The resulting solution was heated in boiling water for 10 min. The solvent was evaporated to dryness and the crude residue was purified by HPLC Method 1. Retention time for the product was at 24 min. Yield: ~ 35%. ¹H NMR (D₂O) δ: 7.87 (m, 5H, Ph), 5.21 (s, 2H, H₂CPh), 4.26-4.1 (m, 3H, HC(NH)CO₂(Glu), HC(NH)CO₂(Lys), HC(NH)CO₂(Lys-linker)), 3.45 -3.18 (bm, 28H, H₂CCO₂, N(CH₂)₂N(DOTA), H₂CNH(Lys), H₂CNH(Lys-linker)), 2.42(m, 2H, H₂C-linker), 2.20 (m, 2H, H₂CCO₂(Glu), 2.06 (m, 3H, H₂C-linker, H₂CNH(Glu)), 1.85-1.18 (m, 21H, H₂CNH(Glu), H₂C(Lys), H₂C(Lys-linker), (CH₂)₄ (linker)). ¹³C (D₂O) δ: 178.2 (CO₂H), 178.1 (CO₂H), 177.9 (CO₂H), 177.5 (CO₂H), 177.4 (CO₂H) 176.3 (CO₂H), 174.5(CO₂CH₂Ph), 173.9, 173.4 (CONH), 160.1,(NHCONH), 135.6 (C, Ph), 129.1(CH, Ph), 128.9 (CH, Ph),128.1(CH, Ph), 67.3 (CH₂Ph), 60.1, 59.6, 57.6, 57.3 (CH₂CO₂H), 53.4 (CH, Glu), 53.2, 53.1(CH, Lys, Lys-linker), 52.9, 52.8, 52.5 (CH₂, DOTA), 39.0, 38.9(CH₂NH, Lys, Lys-CH₂), 35.7, 35.5 (CH₂CO, linker), 31.1 (CH₂CO, Glu), 27.9(CH₂CH(Glu)), 27.7, 27.6, 27.5, 26.4, 25.1(linker, CH₂(Lys), CH₂(Lys-linker), 22.3, 22.2 (CH₂(Lys), CH₂(Lys-linker)). ESIMS m/Z: 1146[M+H]⁺, HRESI⁺-MS: Calcd. for C₄₉H₇₅GaN₉O₁₈, 1146.4486 [M+H], found: 1146.4480.

2-[3-(1-Carboxy-5-{7-[5-carboxy-5-(3-phenyl-2-{3-phenyl-2-[2-(4,7,10-tris-carboxymethyl-1,4,7,10tetraaza-cyclododec-1-yl)-acetylamino]-propionylamino}-propionylamino)-pentylcarbamoyl]-heptanoylamino}-pentyl)-ureido]-pentanedioic acid, 6

Fmoc-Lys(Boc)-Wang resin (100 mg, 0.43 mM) was allowed to swell with CH₂Cl₂ (3 mL) followed by DMF (3 mL). A solution of 20% piperidine in DMF (3 × 3 mL) was added to the resin that was then shaken gently on a mechanical shaker for 30 min at ambient temperature. The resin was washed with DMF (3 × 3 mL) and CH₂Cl₂ (3 × 3 mL). Formation of free amine was assessed by the Kaiser test.⁴² After swelling the resin in DMF, a solution of Fmoc-Phe-OH (3 eq), HBTU (3 eq), HOBt (3 eq), and DIPEA (4.0 eq) in DMF was added and gently shaken for 2 h. The resin was then washed with DMF (3 × 3 mL) and CH₂Cl₂ (3 × 3 mL). The coupling efficiency was assessed by the Kaiser Test. That aforementioned sequence was repeated for two more coupling steps with Fmoc-Phe-OH and DOTA-(t-butyl ester)₃-CO₂H. Final compound was cleaved from the resin using TFA:CH₂Cl₂ (1:1) and concentrated under vacuum to produce 4. The concentrated product was purified by using a C18 SepPak Vac 2g column. The product was eluted with a solution 70/30 water/acetonitrile (0.1% TFA in each). ¹H NMR (D₂O, δ): 7.14-7.00 (m, 10H, Ph), 4.51(m, 1H, HC(Phe)), 4.42 (m, 1H, HC(Phe)), 4.04(m, 1H, HC(Lys)), 3.16-2.4(bm, 30H, H₂CCO₂, N(CH₂)₂N(DOTA), H₂CPh(Phe), H₂CNH(Lys)), 1.61-1.39(m, 4H, H₂C (Lys)), 1.16(m, 2H, H₂C(Lys)). ¹³C (D₂O) δ: 174.8 (CO₂H), 172.24 (CONH), 172 (CONH), 136.5 (C, Phe), 135.8 (C, Phe), 129.3 (CH, Phe), 128.5 (CH, Phe), 126.9 (CH, Phe), 54.6 (CH₂CO₂), 53.07 (CH, Phe, Lys), 52.1-51.0 (CH₂, DOTA), 39.06 (CH₂NH₂(Lys), 36.32 (CH₂Ph), 29.61 (CH₂, Lys), 26.0 (CH₂, Lys), 21.73, (CH₂, Lys). ESIMS:827 [M+1]⁺.

Lyophilized 4 (10 mg, 12 µmol in 2 mL DMF) was added to 5¹⁹ (20 mg, 21.4 µmol in 1 mL DMF) followed by TEA (214 µmol, 30 µL) and then stirred at 25°C for 16 h. After solvent removal, solid residue was treated with 3 mL TFA:CH₂Cl₂ to remove the PMB group. The residue was washed 2 × 5 mL CH₂Cl₂ to remove impurities. The colorless solid residue, compound 6 thus obtained was purified by a C18 SepPak Vac 2g column using an eluent of 70/30 water/acetonitrile (0.1% TFA in each). The product was further purified using preparative RP-HPLC by Method 1, retention time 17 min. Yield: ~ 30%. ¹H NMR (CD₃CO₂D) δ: 7.35-7.20 (m, 10H, Ph), 4.86 (bm, 2H, HC(Phe)), 4.57-4.46 (3H, HC(NH)CO₂(Glu), HC(NH)CO(Lys), HC(NH)CO(Lys-linker)), 4.4-3.0 (m, 30H, H₂CCO₂, N(CH₂)₂N(DOTA), H₂CPh(Phe), H₂CNH(Lys), H₂CNH(Lys-linker)), 2.8(m, 2H, H₂CPh(Phe)), 2.6 (m, 2H, H₂CCO₂(Glu)), 2.3 (m, 5H, H₂CCHNH(Glu), H₂CCONH-linker)), 2.1-1.3 (m, 21H, H₂CCHNH(Glu), (CH₂)₄-linker, (CH₂)₃(Lys), (CH₂)₃(Lys-linker)). ¹³C (CD₃CO₂D) δ: 178.71, (CO₂H), 178.14 (CO₂H), 177.72 (CO₂H), 177.66 (CO₂H), 177.06 (CO₂H), 174.24 (CONH), 173.9(CONH), 161.3(NHCONH), 138.6(C, Ph) 137.7(C, Ph), 130.5 (CH, Ph), 129.5 (CH, Ph), 127.9 (CH, Ph), 127.7(CH, Ph), 56.72 (CH₂CO₂), 56.16 (CH, Phe), 54.6 (CH, Glu), 53.5 (CH, Lys, Lys-linker), 53.3 (CH₂, DOTA), 40.8 (CH₂NH (Lys)), 39.4 (CH₂NH, (Lys-linker)), 37.5 (CH₂Phe), 32.6 (CH₂, (linker)) 31.8 (CH₂, (linker)), 30.7, 29.42, 27.9, 26.53 (CH₂ (linker), CH₂(Lys)). ESIMS m/Z: 1284[M+H]⁺, HRESI⁺-MS: Calcd. for C₆₈H₉₀N₁₁O₂₀, 1284.6365 [M+H], found: 1284.6358.

This compound was prepared according to the same general procedure as described for [^69/71Ga]3. Compound [^69/71Ga]6 was purified by Method 1, retention time 22 min. Yield: ~ 30%. ¹H NMR (MeOD) δ: 7.30-7.20 (m, 10H, Ph), 4.76-4.67(bm, 2H, HC(Phe)), 4.36-4.27 (3H, HC(NH)CO₂(Glu), HC(NH)CO₂(Lys), HC(NH)CO(Lys-linker)), 4.0-3.35 (m, 24H, H₂CCO₂, N(CH₂)₂N(DOTA)), 3.29-3.1(m, 5H, H₂CPh(Phe), H₂CNH(Lys), H₂CNH(Lys-linker)), 3.05(m, 1H, H₂CNH(Lys)), 2.27(m, 2H, H₂CPh(Phe)), 2.4 (m, 2H, H₂CCONH-linker), 2.28-2.1 (m, 5H, H₂CCO₂(Glu), H₂CCHNH(Glu), H₂CCONH-linker)), 1.98-1.8(3H, H₂CCHNH(Glu), CH₂-linker), 1.8-1.3 (m, 18H, (CH₂)₄-linker, (CH₂)₃-Lys, (CH₂)₃-Lys-linker)). ¹³C (MeOD) δ: 175.71 (CO₂H), 174.4 (CO₂H), 174.2 (CO₂H), 173.2 (CO₂H), 171.9 (CO₂H), 170.4(CONH), 170.3, 170.2, 169.9 (CONH), 169.5(CONH), 159.0(NHCONH), 137.3(C, Ph) 136.9(C, Ph), 129.3 (CH, Ph), 129.2 (CH, Ph), 128.3 (CH, Ph), 128.2(CH, Ph), 126.3, 126.2(CH, Ph), 61.8, 60.7, 59.4, 59.3 (CH₂CO₂), 57.6 (CH, (Glu)), 57.5(CH, (Lys), (Lys-linker)), 54.4, 54.3(CH(Phe)), 54.2, 54.1, 52.8, 52.5, 52.3 (CH₂, DOTA), 37.5, 37.4 (CH₂NH, (Lys-linker), Lys), 35.5 (CH₂Phe), 35.4 (CH₂Phe), 32.0 (CH₂CO₂, Glu), 30.8 (CH₂CONH, linker), 29.7 (CH₂CH, Glu), 29.42, 29.3, 29, 7, 27.9, 26.53, 22.5, 22.3 (CH₂(linker), CH₂(Lys), CH₂(Lys-linker)). ESIMS m/Z: 1351[M+H]⁺, HRESI⁺-MS: Calcd. for C₆₈H₈₆GaN₁₁NaO₂₀, 1372.5204 [M+Na]⁺, found: 1372.5199.

Preparation of ⁶⁸Ga

⁶⁸Ga labeling of compounds [⁶⁸Ga]3 and [⁶⁸Ga]6 were performed according to a literature procedure.²⁴ A detailed description for [⁶⁸Ga]3 is given below.

Preconcentration of [⁶⁸Ga(III)]

488 MBq (13 mCi) of ⁶⁸GaCl₃ in 7 mL of 0.1 N HCl were obtained from an 18-month-old 1,850 MBq (50 mCi) ⁶⁸Ge/⁶⁸Ga generator, Eckert-Ziegler (Berlin). The solution was transferred on a cation-exchange cartridge, Phenomenex Strata-X-C (33 µm strong cation exchange resin, part no. 8B-S029-TAK, 30 mg/1mL). The column was eluted with 5 mL of a solution of 20/80 of hydrochloric acid (0.10 N)/acetone. The eluent remaining on the cation exchanger was removed by passage of nitrogen. That process was performed to remove most of the remaining chemical and radiochemical impurities from the resin, whereas ⁶⁸Ga(III) should remain on the column. The column was filled with 150 µL of a 2.4/97.6 HCl (0.05 N)/acetone solution. About 2 min standing appeared to be best for complete desorption of the ⁶⁸Ga(III) from the resin into the liquid phase. An additional 250 µL of that 2.4/97.6 HCl (0.05 N)/acetone solution was applied, and the purified ⁶⁸Ga(III) was obtained in a total volume of 400 µL.

General Radiolabeling Procedure

The 400 µL combined fractions of ⁶⁸Ga(III) in HCl/acetone was used directly for the radiolabeling of 3/6. The concentrated radioactivity was added to 500 µL of deionized H₂O in a standard glass reagent vial containing 100 µl (92 nmol, 1 mg/mL solution) of ligand. No buffer solution was added. The reaction vial was heated at 95°C for 10 min. The complexation was monitored by injecting aliquots of 100 µL (7.77 MBq) of the solution onto the HPLC. Product obtained = 5.92 MBq. For [⁶⁸Ga]3, radiochemical yield: 76.2% (without decay correction) and the radiochemical purity was >99%. HPLC was performed by Method 1 as described in the General experimental section. R_t = 25 min for the desired product and R_t = 19 min for the free ligand. For [⁶⁸Ga]6, radiochemical yield: 70% and radiochemical purity > 99%. HPLC was performed by Method 2 as mentioned in General experimental section. R_t = 22.5 min for the desired product and R_t = 16 min for the free ligand. The acidic eluate was neutralized with 100 µL 0.1M NaHCO₃ solution and the volume of the eluate was reduced under vacuum to dryness. The solid residue was diluted with saline to the desired radioactivity concentration for biodistribution and imaging studies.

Lipophilicity Determination

Partition coefficients, log_o/w (pH = 7.4) values were determined according to a literature procedure.²⁵ Briefly, a solution of either [⁶⁸Ga]3 or [⁶⁸Ga]6 was added to a presaturated solution of 1-octanol (5 mL) mixed with phosphate buffered saline (PBS) (5 mL) in a 15 mL centrifuge tube. After vigorously shaking the mixture, it was centrifuged at 3,000 rpm for 5 min. Aliquots (100 µL) were removed from the two phases and the radioactivity was measured in a γ-counter, 1282 Compugamma CS (LKB, Wallac, Turku, Finland).

Cell Lines and Tumor Models

PC-3 PIP (PSMA+) and PC-3 flu (PSMA−) cell lines were obtained from Dr. Warren Heston (Cleveland Clinic) and were maintained as previously described.¹³ LNCaP cells were obtained from American Type Culture Collection (ATCC, Manassas, VA) and were maintained as per ATCC guidelines. All cells were grown to 80–90% confluence before trypsinization and formulation in Hank’s Balanced Salt Solution (HBSS, Sigma, St. Louis, MO) for implantation into mice.

Animal studies were undertaken in compliance with institutional guidelines related to the conduct of animal experiments. For biodistribution studies of [⁶⁸Ga]3, and [⁶⁸Ga]6 and imaging studies of [⁶⁸Ga]3, male SCID mice (NCI) were implanted subcutaneously with 1 – 5 × 10⁶ PSMA+ PC-3 PIP and PSMA− PC-3 flu cells behind either shoulder. For imaging studies of [⁶⁸Ga]3, male SCID mice (NCI) were implanted subcutaneously with 5 × 10⁶ LNCaP cells behind the right shoulder. Mice were imaged or used in biodistribution studies when the tumor xenografts reached 3 – 5 mm in diameter.

Biodistribution

PSMA+ PC-3 PIP and PSMA− PC-3 flu xenograft-bearing SCID mice were injected via the tail vein with 30 µCi (1.1 MBq) of [⁶⁸Ga]3 or [⁶⁸Ga]6. In case each four mice were sacrificed by cervical dislocation at 30, 60, 120, 180 min pi. for [⁶⁸Ga]3 and at 5, 60, 120, 180 min pi for [⁶⁸Ga]6. The heart, lungs, liver, stomach, pancreas, spleen, fat, kidney, muscle, small and large intestines, urinary bladder, and PC-3 PIP and flu tumors were quickly removed. A 0.1 mL sample of blood was also collected. Each organ was weighed, and the tissue radioactivity was measured with an automated gamma counter (1282 Compugamma CS, Pharmacia/LKB Nuclear, Inc., Gaithersburg, MD). The %ID/g was calculated by comparison with samples of a standard dilution of the initial dose. All measurements were corrected for decay.

PET and CT Imaging

A single SCID mouse implanted with a PSMA+ LNCaP xenograft was injected intravenously with 0.2 mCi (7.4 MBq) of [⁶⁸Ga]3 in 200 µL 0.9% NaCl. At 0.5 h pi, the mouse was anesthetized with 3% isoflurane in oxygen for induction and maintained under 1.5% isoflurane in oxygen at a flow rate of 0.8 L/min. The mouse was positioned in the prone position on the gantry of a GE eXplore VISTA small animal PET scanner (GE Healthcare, Milwaukee, WI). Image acquisition was performed using the following protocol: The images were acquired as a pseudodynamic scan, i.e., a sequence of successive whole-body images were acquired in three bed positions for a total of 120 min. The dwell time at each position was 5 min, such that a given bed position (or mouse organ) was revisited every 15 min. An energy window of 250 – 700 keV was used. Images were reconstructed using the FORE/2D-OSEM method (two iterations, 16 subsets) and included correction for radioactive decay, scanner dead time, and scattered radiation. After PET imaging, the mobile mouse holder was placed on the gantry of an X-SPECT (Gamma Medica Ideas, Northridge, CA) small animal imaging device to acquire the corresponding CT. Animals were scanned over a 4.6 cm field-of-view using a 600 µA, 50 kV beam. The PET and CT data were then co-registered using Amira 5.2.0 software (Visage Imaging Inc., Carlsbad, CA).

Imaging studies and blocking studies of [⁶⁸Ga]6 and [⁶⁸Ga]3 were carried out on PSMA+ PC-3 PIP and PSMA− PC-3 flu xenograft-bearing SCID mice or PSMA+ PC-3 PIP (25.9 MBq in 100 µL NaCl) xenograft-bearing SCID mice. At 30 min, 1 h and 2 h pi the mice were anesthetized and whole-body images were obtained using the PET scanner as mentioned above, in two bed positions, 15 min at each position for a total of 30 min using the same energy window. Images were reconstructed and co-registered with the corresponding CT images using the same methods as described above.

Supplementary Material

Supporting info

Click here to view.^{(436K, pdf)}

Acknowledgments

We thank Dr. Richard L. Wahl for provision of the ⁶⁸Ga generator. We also thank NIH R01 CA134675 and U24 CA92871 and the AdMeTech Foundation for financial support.

Abbreviations


PSMA	prostate-specific membrane antigen

DCFBC	N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-(S)-4-fluorobenzyl-L-cysteine

PMPA	2-(phosphonomethyl)pentanedioic acid

DOTA	1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid

PET	positron emission tomography

Footnotes

Supporting Information Available

Additional figures demonstrating the HPLC traces for [⁶⁸Ga]3 and [⁶⁸Ga]6 as well as the high-resolution mass spectra for these compounds are provided. Also provided is a figure containing a PET blocking study for [⁶⁸Ga]3. These materials are available free of charge via the Internet at http://pubs.acs.org.

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