Synthesis and Evaluation of 64Cu-Labeled Monomeric and Dimeric

Nov 28, 2012 - Department of Nuclear Medicine, Xijing Hospital, The Fourth Military Medical University, Xi'an, Shaanxi, China, 710032. ABSTRACT: The ...
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Synthesis and Evaluation of 64Cu-Labeled Monomeric and Dimeric NGR Peptides for MicroPET Imaging of CD13 Receptor Expression Kai Chen,*,† Wenhui Ma,‡ Guoquan Li,†,‡ Jing Wang,*,‡ Weidong Yang,‡ Li-Peng Yap,† Lindsey D. Hughes,† Ryan Park,† and Peter S. Conti*,† †

Molecular Imaging Center, Department of Radiology, Keck School of Medicine, University of Southern California, Los Angeles, California 90033, United States ‡ Department of Nuclear Medicine, Xijing Hospital, The Fourth Military Medical University, Xi’an, Shaanxi, China, 710032 ABSTRACT: The NGR-containing peptides have been shown to bind specifically to CD13/aminopeptidase N (APN) receptor, one of the attractive tumor vasculature biomarkers. In this study, we evaluated 64 Cu-labeled monomeric and dimeric NGR peptides for microPET imaging of CD13 receptor expression in vivo. Western blot analysis and immunofluorescence staining were performed to identify CD13-positive and CD13-negative cell lines. NGR-containing peptides were conjugated with 1,4,7,10-tetraazadodecane-N,N′,N″,N‴-tetraacetic acid (DOTA) and labeled with 64Cu (t1/2 = 12.7 h) in ammonium acetate buffer. The resulting monomeric (64Cu-DOTA−NGR1) and dimeric (64Cu-DOTA−NGR2) peptides were then subjected to in vitro stability, cell uptake and efflux, small animal micorPET, and biodistribution studies. In vitro studies demonstrated that CD13 receptors are overexpressed in human fibrosarcoma HT-1080 cells and negative in human colon adenocarcinoma HT-29 cells. The binding affinity of 64Cu-DOTA−NGR2 to HT-1080 cells was measured to be within low nanomolar range and about 2-fold higher than that of 64Cu-DOTA−NGR1. For small animal microPET studies, 64CuDOTA−NGR2 displayed more favorable in vivo performance in terms of higher tumor uptake and slower tumor washout in CD13-positive HT-1080 tumor xenografts as compared to 64Cu-DOTA−NGR1. As expected, significantly lower tumor uptake and poorer tumor/normal organ contrast were observed for both 64Cu-DOTA−NGR1 and 64Cu-DOTA−NGR2 in CD13negative HT-29 tumor xenografts in comparison with those in the HT-1080 tumor xenografts. The CD13-specific tumor activity accumulation of both 64Cu-DOTA−NGR1 and 64Cu-DOTA−NGR2 was further demonstrated by significant reduction of tumor uptake in HT-1080 tumor xenografts with a coinjected blocking dose of cyclic NGR peptide [c(CNGRC)]. The biodistribution results were consistent with the quantitative analysis of microPET imaging. We concluded that both 64Cu-DOTA−NGR1 and 64 Cu-DOTA−NGR2 have good and specific tumor uptake in CD13-positive HT-1080 tumor xenografts. 64Cu-DOTA−NGR2 showed higher tumor uptake and better tumor retention than 64Cu-DOTA−NGR1, presumably due to bivalency effect and increase in apparent molecular size. 64Cu-DOTA−NGR2 is a promising PET probe for noninvasive detection of CD13 receptor expression in vivo. KEYWORDS: microPET imaging, NGR peptide, CD13, tumor angiogenesis, bivalency effect, 64Cu labeling

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INTRODUCTION Angiogenesis, the formation of new blood vessels from preexisting vasculature, is a fundamental process occurring during tumor progression.1,2 A number of studies suggest that tumorvasculature formation is a complex multistep process that follows a characteristic sequence of events mediated and controlled by growth factors, adhesion molecules, and cellular receptors.3−5 CD13 [also referred to as aminopeptidase N (APN)] receptor is a zinc dependent membrane-bound ectopeptidase, which has been identified as a critical regulator of angiogenesis6 where its expression on activated blood vessels is induced by angiogenic signals.7 To date, CD13 receptor has been found to be a surface marker for malignant myeloid cells,8,9 and reach high levels of expression in association with the progression of tumors, including prostate, lung, and ovarian cancer.10−12 Because CD13 receptor plays a vital role in tumor angiogenesis, it is important to develop an approach for © 2012 American Chemical Society

noninvasively imaging CD13 receptor levels in living subjects, which would allow us to early detect CD13-targeted tumor angiogenesis as well as effectively monitor response to antitumor vasculature therapy. Positron emission tomography (PET) offers the opportunity of noninvasive quantification of diseases associated with biochemical processes.13 Compared to morphological imaging techniques, such as computed tomography, PET requires the injection of molecular probes in a tested subject in order to acquire the imaging signal from molecular probes labeled with positron-emitting radionuclides.14,15 Numerous targeting moieties have been employed as vehicles of PET probes, including Received: Revised: Accepted: Published: 417

October 4, 2012 November 6, 2012 November 28, 2012 November 28, 2012 dx.doi.org/10.1021/mp3005676 | Mol. Pharmaceutics 2013, 10, 417−427

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labeled NGR monomer and dimer were investigated. The ability of using newly developed 64Cu-labeled NGR peptides to image CD13 receptor expression in vivo by microPET was evaluated in subcutaneous CD13-positive HT-1080 fibrosarcoma xenografts using female athymic nude mice. CD13-specific targeting of both 64Cu-labeled NGR monomer and dimer was further assessed in CD13-negative mouse HT-29 colon adenocarcinoma xenografts and a blocking study in mouse HT-1080 tumor xenografts.

small molecules, peptides, protein, antibody and its fragments, and nanoparticles.15,16 Among these moieties, low-molecularweight small peptides demonstrate a number of distinct advantages over others, including favorable pharmacokinetic and tissue distribution patterns, good permeability properties, low toxicity and immunogenicity, and flexibility in chemical modification and radiolabeling.16 However, identification of target-specific peptide is far from trivial. Screening of bacteriophage (phage) display libraries is emerging as a powerful technique for providing a means to improve peptide affinity and generate unique peptides that bind any given target.17,18 Through in vivo screening of a phage-displayed peptide library, a tumor vasculature homing phage carrying sequence CNGRCVSGCAGRC was selected by using human breast carcinoma xenografts.19 Tumor homing of this sequence can be inhibited by the coinjection with CNGRC peptide, indicating that this short cyclic loop is a functional tumor targeting peptide. Various NGR containing motifs, including linear NGR, such as NGRAHA and GNGRG, and cyclic NGR, such as CNGRC and CVLNGRMEC, were then identified20,21 as tumor vasculature homing phages by screening in vitro and in vivo phage libraries, suggesting that the NGR-containing peptide may be a specific ligand for binding to a molecular target associated with tumor angiogenesis. Until 2000, the molecular basis behind NGR tumor-homing properties was revealed, and CD13 receptor was identified as the molecular target of NGR-containing peptide.22 Since then, a number of NGR-containing derivatives have been developed for both CD13-targeted tumor imaging and therapy.23−26 Although most of the NGR peptide-based probes could target tumors by binding to CD13 receptors, they usually suffer from modest tumor uptake and unfavorable pharmacokinetics which limit their applications for in vivo imaging and therapy.25,26 In order to enhance the binding affinity to a presumed receptor and, thus, achieve higher receptor-targeted tumor uptake, multimerization of a ligand has been proved to be an effective approach in the development of imaging probes and chemotherapeutics.15,27−33 For instance, others have reported that dimeric RGD peptide with two repeating cyclic RGD units significantly enhanced the binding affinity of RGD ligand to integrin αvβ3 receptor due to bivalency effect.34−36 Because CD13 receptor is a cell-surface target of tumor angiogenesis similar to integrin αvβ3 receptor, we hypothesized that the receptor binding of one NGR peptide may significantly enhance the “local concentration” of the other NGR peptides in the vicinity of the receptor, leading to a faster rate of receptor binding or a slower rate of dissociation of NGR multimers from the CD13 receptors, and resulting in higher uptake and longer retention time in the tumor. A dimeric NGR peptide with two repeating cyclic NGR units is thus expected to enhance the affinity of the receptor−ligand interactions through a bivalency effect. The apparent increase in molecular size may also prolong circulation time of the dimer and consequently reduce tumor washout rate. In this study we applied the bivalency principle and developed a novel dimeric NGR peptide using glutamate as a branching linker. The resulting NGR peptide dimer was conjugated with a commercially available macrocylic chelator: 1,4,7,10-tetraazadodecane-N,N′,N″,N‴-tetraacetic acid (DOTA) and labeled with 64Cu. 64Cu-Labeled NGR monomer was also prepared as a comparison. The in vitro stability, lipophilicity, and tumor cell uptake and retention of both 64Cu-

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EXPERIMENTAL SECTION General. All chemicals (reagent grade) were obtained from commercial suppliers and used without further purification. The NGR peptide [GGGCNGRC; disulfide Cys:Cyc = 4−8] and Boc-protected Gly3 peptide were purchased from C S Bio Company, Inc. (Menlo Park, CA, USA). DOTA-NHS ester was purchased from Macrocyclics Inc. (Dallas, TX, USA). [64Cu]CuCl2 was purchased from the Washington University in St. Louis, MO, USA. Water was purified using a Milli-Q ultrapure water system from Millipore (Milford, MA, USA), followed by passing through a Chelex 100 resin before bioconjugation and radiolabeling. Mass spectra were obtained on a ThermoElectron Finnigan LTQ mass spectrometer equipped with an electrospray ionization source (Thermo Scientific, USA). HPLC Methods. Analytic and semipreparative reversed phase HPLC were accomplished on two Waters 515 HPLC pumps, a Waters 2487 absorbance UV detector, and a Ludlum model 2200 radioactivity detector, which were operated by Waters Empower 2 software. The purification of DOTAconjugated NGR peptides was performed on a Phenomenex Luna C18 reversed phase column (5 μm, 250 × 10 mm). The flow rate was 4 mL/min for semipreparative HPLC, with the mobile phase starting from 95% solvent A (0.1% TFA in water) and 5% solvent B (0.1% TFA in acetonitrile) to 40% solvent A and 60% solvent B at 27.5 min. The UV absorbance was monitored at 214 and 254 nm. The analytic HPLC and 64Cu labeling purification were performed on a Phenomenex Luna C18 reversed phase analytical column (5 μm, 250 × 4.6 mm). The flow rate was 1 mL/min with the mobile phase starting from 100% solvent A (0.1% TFA in water) to 50% solvent A and 50% solvent B (0.1% TFA in acetonitrile) at 22.5 min. Synthesis of DOTA−NGR1 Peptide. The NGR peptide [GGGCNGRC; disulfide Cys:Cyc = 4−8] (3.0 mg, 4.16 μmol) dissolved in 0.5 mL of DMF was mixed with DOTA-NHS ester (3.8 mg, 5.0 μmol) and DIPEA (25 μL). After sonication at 40 °C for 2 h, the reaction was quenched by adding 50 μL of 5% acetic acid (HOAc). The mixture was dissolved in water and purified by analytic HPLC. The peak containing the DOTA− NGR1 peptide (tR = 15.5 min) was collected and lyophilized to afford a fluffy white powder (3.9 mg, yield: 85%). ESI-MS m/z C40H66N16O17S2 [M + H]+ calcd, 1107.42; found, 1107.90. Synthesis of Boc-Protected NGR2 Peptide. Bocprotected glutamic acid activated ester Boc-E(OSu)2 was prepared according to a procedure described previously.33 To a solution of the NGR peptide [GGGCNGRC; disulfide Cys:Cyc = 4−8] (10.0 mg, 14 μmol) in sodium borate buffer (pH 8.5) was added Boc-E(OSu)2 (2.05 mg, 4.6 μmol) in anhydrous DMSO (25 μL). The reaction was stirred at room temperature for 2 h, and the desired product was isolated by semipreparative HPLC (tR = 13.2 min). The collected fractions were combined and lyophilized to afford a fluffy white powder (5.1 mg, yield: 67%). ESI-MS: m/z C58H93N25O24S4 [M + H]+ calcd, 1652.57; found, 1652.65. 418

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Synthesis of NGR2 Peptide. Boc-protected NGR2 peptide (5.0 mg, 3 μmol) was dissolved in 0.5 mL of TFA:triisopropylsilane:water (95:2.5:2.5) solution. The mixture was stirred at room temperature for 1 h. After evaporation of solvent, the residue was redissolved in 0.5 mL of water and purified by semipreparative HPLC. The peak containing the NGR2 peptide was collected (tR = 10.3 min) and lyophilized to afford a fluffy white powder (4.1 mg, yield: 88%). ESI-MS: m/z C53H85N25O22S4 [M + H]+ calcd, 1552.52; found, 1552.59. Synthesis of Boc-Protected Gly3-NGR2 Peptide. Bocprotected Gly3 peptide [BocNH-Gly3-OH] (0.7 mg, 2.42 μmol) was activated by HATU (0.92 mg, 2.42 μmol) and HOAt (0.33 mg, 2.42 μmol) in 200 μL of DMF for 30 min. To the mixture was added NGR2 peptide (2.5 mg, 1.6 μmol) dissolved in 200 μL of DMF. The mixture was adjusted to pH 8.5 with DIPEA and sonicated at room temperature for 2 h. The crude peptide was purified by semipreparative HPLC. The peak containing the desired product was collected (tR = 14.0 min) and lyophilized to afford a fluffy white powder (2.1 mg, yield: 72%). ESI-MS: m/z C64H102N28O27S4 [M + H]+ calcd, 1823.64; found, 1823.90. Synthesis of Gly3-NGR2 Peptide. Boc-protected Gly3NGR2 peptide (2.0 mg, 1.1 μmol) was dissolved in 0.25 mL of TFA:triisopropylsilane:water (95:2.5:2.5) solution. The mixture was stirred at room temperature for 1 h. After evaporation of solvent, the residue was redissolved in 0.5 mL of water and purified by semipreparative HPLC. The peak containing the Gly3-NGR2 peptide was collected (tR = 10.9 min) and lyophilized to afford a fluffy white powder (1.6 mg, yield: 85%). ESI-MS: m/z C59H94N28O25S4 [M + H]+ calcd, 1723.58; found, 1723.80. Synthesis of DOTA−NGR2 Peptide. Gly3-NGR2 peptide (1.5 mg, 0.87 μmol) dissolved in 0.5 mL of DMF was mixed with DOTA-NHS ester (1.07 mg, 1.04 μmol) and DIPEA (20 μL). After sonication at 40 °C for 2 h, the reaction was quenched by adding 50 μL of 5% acetic acid (HOAc). The solvent was removed in vacuo. The resulting residue was dissolved in water and purified by analytical HPLC. The peak containing the DOTA−NGR2 peptide was collected (tR = 16.6 min) and lyophilized to afford a fluffy white powder (1.56 mg, yield: 85%). ESI-MS: m/z C75H120N32O32S4 [M + 2H]2+ calcd, 1057.38; found, 1057.70. 64 Cu Labeling and Formulation. [64Cu]Cu(OAc)2 was prepared by adding 37−111 MBq of [64Cu]CuCl2 in 0.1 N HCl into 300 μL of 0.4 M ammonium acetate buffer (pH = 5.5), followed by mixing and incubating for 15 min at room temperature. The [64Cu]Cu(OAc)2 solution (37−111 MBq) was then added into a solution of DOTA−NGR1 or DOTA− NGR2 peptide (5 μg of peptide per mCi of 64Cu) dissolved in 0.4 M NH4OAc (pH = 5.5) solution. The reaction mixture was incubated at 40 °C for 30 min and purified by analytical HPLC. The radioactive peak containing 64Cu-DOTA−NGR1 or 64CuDOTA−NGR2 peptide was collected and concentrated by rotary evaporation. The product was then reconstituted in 500−800 μL of PBS, and passed through a 0.22 μm Millipore filter into a sterile dose vial for use in the following experiments. Partition Coefficient. The partition coefficient value was expressed as log P. Log P of 64Cu-DOTA−NGR1 or 64CuDOTA−NGR2 peptide was determined by measuring the distribution of radioactivity in 1-octanol and PBS. Approximately 185 kBq of 64Cu-DOTA−NGR1 or 64Cu-DOTA− NGR2 peptide in 2 μL of PBS (pH = 7.4) was added to a vial containing 0.5 mL of 1-octanol and 0.5 mL of PBS (pH = 7.4).

After vigorously vortexing for 10 min, the vial was centrifuged at 12,500 rpm for 5 min to ensure the complete separation of layers. 200 μL of each layer was pipetted into test tubes, and radioactivity was measured using a gamma counter (PerkinElmer Packard Cobra). The mean value was calculated from triplicate experiments. In Vitro Stability Determination. The stability of 64CuDOTA−NGR1 or 64Cu-DOTA−NGR2 was tested in PBS and mouse serum. In brief, 3.7 MBq of the 64Cu-DOTA−NGR1 or 64 Cu-DOTA−NGR2 was pipetted into 0.5 mL of the PBS and incubated in PBS at room temperature or mouse serum at 37 °C with gentle shaking at 300 rpm. For PBS study, at various time points (1, 6, and 24 h), an aliquot of the solution was directly taken and the radiochemical purity was determined by reverse-phase HPLC under identical conditions. For mouse serum study, at various time points (1, 6, and 24 h), trifluoroacetic acid was added, and the soluble fraction was clarified with a 0.22 mm filter. An aliquot of the solution was then taken, and the radiochemical purity was determined by reverse-phase HPLC under identical conditions. Cell Culture. The human fibrosarcoma cell line HT-1080 was obtained from the American Type Culture Collection (Manassas, VA, USA) and was maintained at 37 °C in a humidified atmosphere containing 5% CO2 in Dulbecco’s modified medium and supplemented with 10% fetal bovine serum (Life Technologies, Grand Island, NY, USA). The human colon adenocarcinoma HT-29 cell line, obtained from the American Type Culture Collection, was grown in RPMI1640 medium supplemented with 10% fetal bovine serum in an atmosphere containing 5% CO2 at 37 °C. Western Blot Analysis. HT1080 or HT-29 cells grown in a 75 cm2 culture flask were suspended in lysis buffer (Beyotime, China) supplemented with complete, mini protease inhibitors (Roche, Canada). The cell debris was then removed by centrifugation (10 000 rpm at 4 °C for 10 min), and the protein concentration was determined with the Bradford Protein Assay Kit (Beyotime, China). Samples of cell extracts containing 40 μg of protein were loaded on SDS−PAGE gels and transferred to polyvinylidene fluoride membrane filters (Life Technologies, Grand Island, NY, USA). CD13 protein was detected with antiCD13 antibody (1:100, Santa Cruz Biotechnology, CA, USA) and peroxidase-conjugated secondary antibody (1:400, Life Technologies, Grand Island, NY, USA). The antigen−antibody complexes on the membranes were visualized with ECL Western Blotting Detection System (Thermo Scientific, NC, USA) with ChemiDOC XRS+ (Bio-Rad, Hercules, CA, USA). Beta-actin was detected with anti-β-actin as an internal loading control. Immunofluorescence Staining. HT-1080 or HT-29 cells were plated into a 24-well plate at a density of 5 × 104 cells/ well. After overnight incubation, cells were fixed with 4% of paraformaldehyde for 10 min, and washed with PBS three times. The cells were then incubated in 3% BSA for 30 min to block nonspecific binding, followed by overnight incubation at 4 °C with anti-CD13 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted to 1:50 in PBS (pH 7.4) containing 1% BSA. On the following day, the cells were washed with PBS three times, and incubated for 45 min with a secondary goat anti-rabbit IgG fluorescein isothiocyanate (FITC) conjugated antibody (1:400, Life Technologies, Grand Island, NY, USA) diluted 1:400 in PBS (pH 7.4) containing 1% BSA. After washing with PBS three times, the cells were mounted in a mounting medium containing DAPI. The cells 419

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Figure 1. Scheme of the synthesis of 64Cu-DOTA−NGR1 (a) and 64Cu-DOTA−NGR2 (b).

were then visualized by an Olympus IX71 fluorescence microscope (Olympus, Japan). Cell Binding Assay. HT-1080 cells (1 × 106 cells/plate) were plated at a uniform cell density and incubated overnight. The cells were washed twice for 2 min with ice-cold binding buffer (25 mM HEPES and 1% BSA). The cells were then incubated for 1 h with 99mTc-labeled linear NGR peptide (sequence: YGGCNGRC; prepared in house and data not shown), in the presence of various concentrations of DOTA− NGR1 or DOTA−NGR2. After washing with ice-cold binding buffer three times, the cells were lysed in 200 μL of lysis buffer. The cell-associated radioactivity was then measured using a gamma counter. The best-fit 50% inhibitory concentration (IC50) values were calculated by fitting the data with nonlinear regression using Graph-Pad Prism 5.0 (GraphPad Software, CA, USA). Experiments were conducted in triplicate. Cell Uptake and Efflux Studies. The cell uptake and efflux studies were performed as previously described with some modification.37 HT-1080 or HT-29 cells were seeded into a 48well plate at a density of 2.5 × 105 cells per well 24 h prior to the experiment. Tumor cells were then incubated with 64CuDOTA−NGR1 or 64Cu-DOTA−NGR2 peptide (370 kBq/ well) at 37 °C for 15, 30, 60, and 120 min. After incubation, tumor cells were washed three times with ice-cold PBS and harvested by trypsinization with 0.25% trypsin/0.02% EDTA (Life Technologies, Grand Island, NY, USA). At the end of trypsinization, wells were examined under a light microscope to ensure complete detachment of cells. Cell suspensions were collected and measured in a gamma counter (Perkin-Elmer Packard Cobra). Cell uptake data was presented as percentage of total input radioactivity after decay correction. Experiments were performed twice with triplicate wells. For efflux studies,

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Cu-DOTA−NGR1 or 64Cu-DOTA−NGR2 peptide (370 kBq/well) was first incubated with HT-1080 or HT-29 cells in a 48-well plate for 2 h at 37 °C to allow internalization. Cells were then washed twice with PBS and incubated with cell culture medium for 15, 30, 60, and 120 min. After washing three times with PBS, cells were harvested by trypsinization with 0.25% trypsin/0.02% EDTA (Life Technologies, Grand Island, NY, USA). Cell suspensions were collected and measured in a gamma-counter (Perkin-Elmer Packard Cobra). Experiments were conducted twice with triplicate wells. Cell efflux data was presented as percentage of added dose after decay correction. Animal Model. All animal studies were performed according to a protocol approved by the Institutional Animal Care and Use Committee of University of Southern California. Female athymic nude mice (about 4−6 weeks old, with a body weight of 20−25 g) were obtained from Harlan Laboratories, Inc. (Livermore, CA, USA). The HT-1080 human fibrosarcoma xenograft model and the HT-29 human colon adenocarcinoma xenograft model were generated by subcutaneous injection of 5 × 106 tumor cells into the front flank of female athymic nude mice. The tumors were allowed to grow 3−4 weeks until 200− 500 mm3 in volume. Tumor growth was followed by caliper measurements of the perpendicular dimensions. MicroPET Imaging and Blocking Experiment. MicroPET scans and imaging analysis were performed using a rodent scanner (microPET R4 scanner; Siemens Medical Solutions). About 7.4 MBq of 64Cu-DOTA−NGR1 or 64Cu-DOTA− NGR2 peptide was intravenously injected into each mouse under isoflurane anesthesia. Five-minute static scans were acquired at 1, 2, and 4 h pi, and a ten-minute static scan was acquired at 24 h pi. The images were reconstructed by a two420

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dimensional ordered-subsets expectation maximum (OSEM) algorithm. For each microPET scan, regions of interest were drawn over the tumor, normal tissue, and major organs on the decay-corrected whole-body coronal images. The radioactivity concentration (accumulation) within the tumor, muscle, liver, and kidneys was obtained from the mean value within the multiple regions of interest and then converted to % ID/g. For the blocking experiment, mice bearing HT-1080 tumors were scanned (5 min static) at 4 h after the coinjection of 7.4 MBq of 64Cu-DOTA−NGR1 or 64Cu-DOTA−NGR2 with 20 mg/kg NGR peptide [c(CNGRC)] per mouse. Biodistribution Studies. The HT-1080 or HT-29 tumor bearing nude mice (n = 5/group) were injected with 7.4 MBq of 64Cu-DOTA−NGR1 or 64Cu-DOTA−NGR2. At 24 h after injection of the radiolabeled probe, mice were sacrificed and dissected. Blood, tumor, major organs, and tissues were collected and weighed wet. The radioactivity in the tissues was measured using a gamma counter (Perkin-Elmer Packard Cobra). The results were presented as percentage injected dose per gram of tissue (% ID/g). For each mouse, the radioactivity of the tissue samples was calibrated with a known aliquot of the injected activity. Mean uptake (% ID/g) for a group of animals was calculated with standard deviations. Statistical Analysis. Quantitative data were expressed as mean ± SD. Means were compared using one-way ANOVA and Student’s t test. P values 99%. 64Cu-labeled DOTA−NGR1 or DOTA−NGR2 peptide was analyzed and purified by HPLC. The HPLC retention time of 64Cu-DOTA− NGR1 was 15.58 min, while retention time of 64Cu-DOTA− NGR2 was determined to be 16.67 min under identical HPLC conditions (Figure 2). On the analytic HPLC, no significant difference of retention time between 64Cu-labeled probe and unlabeled conjugate was observed. The specific activity of 64CuDOTA−NGR1 or 64Cu-DOTA−NGR2 was estimated to be about 37 MBq/nmol. The 64Cu-labeled probe was used immediately after formulation. Log P Value and in Vitro Stability. The octanol/water partition coefficient (log P) for 64Cu-DOTA−NGR1 and 64CuDOTA−NGR2 was determined to be −2.35 ± 0.03 and −2.54 ± 0.07, respectively, suggesting that both 64Cu-labeled monomeric and dimeric NGR peptides are rather hydrophilic. The in vitro stability of 64Cu-DOTA−NGR1 and 64Cu-DOTA− NGR2 was studied in PBS (pH 7.4) at room temperature or mouse serum at physiological temperature 37 °C for different time intervals (1, 6, and 24 h). The stability was presented as

Figure 3. Stability of 64Cu-DOTA−NGR1 and 64Cu-DOTA−NGR2 in PBS (pH = 7.4) at room temperature and mouse serum at 37 °C for 1, 6, and 24 h.

NGR1 and 64Cu-DOTA−NGR2 showed very good stability in PBS at room temperature as well as in mouse serum at 37 °C. After 24 h of incubation at room temperature, more than 97.6% of 64Cu-DOTA−NGR1 and >97.2% of 64Cu-DOTA−NGR2 remained intact in PBS. For the stability study in mouse serum, after 24 h of incubation at 37 °C, more than 94.5% of 64CuDOTA−NGR1 and >94.0% of 64Cu-DOTA−NGR2 remained as the parent radiolabeled probe. Western Blot and Immunofluorescence Staining. Western blot analysis of CD13 expression in HT-1080 and HT-29 cells is shown in Figure 4a. For HT-1080 cells, there was a clear band at 150 kDa which belongs to CD13 expression, whereas no band was identified at 150 kDa for HT-29 cells, suggesting that CD13 is indeed overexpressed in HT-1080 cells but not in HT-29 cells. In addition, immunofluorescence staining of HT-1080 and HT-29 cells showed that strong green fluorescence signal was observed on the cell membrane of HT1080 cells but not HT-29 cells (Figure 4b), further confirming that HT-1080 is a positive cell line for CD13 expression while the HT-29 cell line is negative. 421

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Cell Uptake and Efflux. Cell uptake and retention of 64CuDOTA−NGR1 and 64Cu-DOTA−NGR1 were examined in CD13-positive HT-1080 and CD13-negative HT-29 tumor cells. The cell uptake study revealed that both 64Cu-DOTA− NGR1 and 64Cu-DOTA−NGR2 bind to CD13-positive HT1080 cells, but not to CD13-negative HT-29 cells. For the monomeric 64Cu-DOTA−NGR1, about 0.78 ± 0.04% of probe uptake in HT-1080 cells was determined during the first hour of incubation. After 2 h incubation, the uptake of 64CuDOTA−NGR1 in HT-1080 cells reached the maximum of 1.00 ± 0.08% of total input radioactivity (Figure 6a left, solid line). The cell efflux study showed that 64Cu-DOTA−NGR1 has good cell retention in HT-1080 cells. During 2 h of cell efflux study, only about 0.4% (from 1.00% to 0.62% of total input radioactivity) of 64Cu-DOTA−NGR1 efflux was determined (Figure 6a right, solid line). In the CD13-negative cells, both cellular uptake and retention of 64Cu-DOTA−NGR1 were obtained at the minimal level of total input radioactivity after 2 h incubation. The values were determined to be 0.27 ± 0.06% for cell uptake and 0.22 ± 0.03% for cell efflux, respectively (Figure 6a, dotted line). In addition, for the dimeric 64CuDOTA−NGR2, about 1.42 ± 0.20% of probe uptake in HT1080 cells was observed after 2 h incubation (Figure 6b left, solid line), which was about 1.4-fold higher than that of monomeric 64Cu-DOTA−NGR1. The cell efflux study showed that 64Cu-DOTA−NGR2 exhibits reasonable cell retention in HT-1080 cells. During 2 h of cell efflux study, about 0.70% (from 1.42% to 0.72% of total input radioactivity) of 64CuDOTA−NGR2 efflux was determined (Figure 6b right, solid line). Similar to the case of 64Cu-DOTA−NGR1 in the CD13negative cells, 64Cu-DOTA−NGR2 demonstrated minimal uptake and efflux properties after 2 h incubation. The values were determined to be 0.44 ± 0.07% for cell uptake and 0.23 ± 0.04% for cell efflux, respectively (Figure 6b, dotted line). MicroPET Imaging. The tumor-targeting efficacy and biodistribution pattern of 64Cu-DOTA−NGR1 and 64CuDOTA−NGR2 were evaluated in nude mice bearing human fibrosarcoma HT-1080 xenografts (n = 5) and human colon adenocarcinoma HT-29 xenografts (n = 5) at multiple time points (1, 2, 4, and 24 h) with static microPET scans. The CD13-positive HT-1080 tumors were all clearly visible with high contrast to contralateral background at all time points measured beginning 1 h after injection of 64Cu-DOTA−NGR1 or 64Cu-DOTA−NGR2; whereas the CD13-negative HT-29 tumors exhibited minimal uptake of 64Cu-DOTA−NGR1 or 64 Cu-DOTA−NGR2. Representative decay-corrected coronal slices that contained the tumor at 4 h pi were shown in Figure 7. Predominant uptake of 64Cu-DOTA−NGR1 and 64CuDOTA−NGR2 was also observed in the liver and kidneys at the early time points for both HT-1080 and HT-29 xenografts. Tumor and major organ activity accumulation in the microPET scans was quantified by measuring the ROIs that encompassed the entire organ on the coronal images. The time−activity curves of HT-1080 tumor, liver, kidneys, and muscle after injection of 64Cu-DOTA−NGR1 or 64Cu-DOTA−NGR2 are depicted in Figure 8a and Figure 8b. For 64Cu-DOTA−NGR1, the HT-1080 tumor uptake was calculated to be 3.33 ± 0.10, 3.09 ± 0.20, 2.32 ± 0.17, and 1.79 ± 0.27% ID/g at 1, 2, 4, and 24 h pi, respectively. As a function of time, radioactivity was steadily cleared from the liver and rapidly excreted from the kidneys. The liver uptake values were calculated to be 8.11 ± 0.56, 7.65 ± 0.76, 6.39 ± 0.41, and 3.90 ± 0.45% ID/g at 1, 2, 4, and 24 h pi, respectively. The kidney uptake values were

Figure 4. (a) Representative Western blot analyses of HT-1080 and HT-29 cell lysates for CD13 receptor expression (150 kDa). Beta-actin was used as a loading control. (b) Immunofluorescence staining of CD13 receptor expression in HT-1080 and HT-29 cells with FITC− goat anti-rabbit IgG (green). The cells were costained with DAPI (blue) for nucleus presentation. Magnification, ×20; scale bar = 20 μm. Both Western blot and immunofluorescence staining data demonstrated that CD13 receptors are overexpressed in HT-1080 cells, but not in HT-29 cells.

Cell-Based Binding Assay. Based on the results from Western blot analysis and immunofluorescence staining, it was clear that the human fibrosarcoma HT-1080 cell line overexpresses CD13/APN receptors. Therefore, we used the HT-1080 cells to measure the CD13 receptor binding affinity of DOTA−NGR1 and DOTA−NGR2 by a competitive cellbinding assay, where a 99mTc-labeled linear NGR peptide was employed as a CD13-specific radioligand for competitive displacement. The IC50 values of DOTA−NGR1 and DOTA−NGR2, which represent their concentrations required to displace 50% of 99mTc-labeled linear NGR peptide bound to the HT-1080 cells, were determined to be 1.27 ± 0.25 nM and 0.62 ± 0.29 nM, respectively (Figure 5). The cell-based binding assay demonstrated that NGR dimer had about 2-fold higher CD13 avidity than the corresponding monomeric NGR peptide.

Figure 5. In vitro inhibition of DOTA−NGR1 and DOTA−NGR2 bound to CD13 on HT-1080 cells by a 99mTc-labeled linear NGR peptide. The IC50 values of DOTA−NGR1 and DOTA−NGR2 were calculated to be 1.27 ± 0.25 nM and 0.62 ± 0.29 nM, respectively. 422

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Figure 6. Cell uptake and efflux assay. (a) Time-dependent cell uptake (left) and efflux (right) of 64Cu-DOTA−NGR1 (n = 3, mean ± SD) using human fibrosarcoma HT-1080 cells (solid line) and human colon adenocarcinoma HT-29 cells (dotted line). (b) Time-dependent cell uptake (left) and efflux (right) of 64Cu-DOTA−NGR2 (n = 3, mean ± SD) using HT-1080 cells (solid line) and HT-29 cells (dotted line).

Figure 7. Representative decay-corrected whole-body microPET images of mice bearing HT-1080 or HT-29 tumors (n = 5/group) on right front flank after intravenous (iv) administration of 7.4 MBq of 64 Cu-DOTA−NGR1 or 64Cu-DOTA−NGR2. The blocking study was performed by iv injection of 7.4 MBq of 64Cu-DOTA−NGR1 or 64CuDOTA−NGR2 with coinjection of 20 mg/kg of NGR peptide [c(CNGRC)] as a blocking agent in HT-1080 tumor xenografts (n = 5/group). MicroPET images are shown at 4 h pi. Tumors are indicated by arrows. Figure 8. Time−activity curves of HT-1080 tumor, liver, kidneys, and muscle from quantitative microPET imaging analysis of 64Cu-DOTA− NGR1 (a) and 64Cu-DOTA−NGR2 (b). ROIs are shown as the mean % ID/g ± SD (n = 5/group).

calculated to be 8.80 ± 0.51, 7.29 ± 0.24, 4.61 ± 0.47, and 0.72 ± 0.21% ID/g at 1, 2, 4, and 24 h pi, respectively. Accumulation of the radiolabeled probe in most other organs (except for intestine) was at a very low level. For 64Cu-DOTA−NGR2, the HT-1080 tumor uptake was calculated to be 6.53 ± 0.20, 6.09 ± 0.18, 5.22 ± 0.17, and 3.60 ± 0.23% ID/g at 1, 2, 4, and 24 h

pi, respectively. The HT-1080 tumor uptake of 64Cu-DOTA− NGR2 was about 2-fold higher than that of 64Cu-DOTA− 423

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Table 1. Decay-Corrected Biodistribution of 64Cu-DOTA−NGR1 and 64Cu-DOTA−NGR2 at 4 h Postinjection in TumorBearing Mice Quantified by MicroPET Imaginga HT-1080 tissue

a

64

Cu-DOTA−NGR1 ± ± ± ±

tumor (T) muscle (M) liver (L) kidneys (K)

2.32 0.59 6.39 4.61

0.17 0.06 0.41 0.47

T/M T/L T/K

3.91 ± 0.19 0.35 ± 0.21 0.52 ± 0.27

64

HT-1080 blockade

Cu-DOTA−NGR2 5.22 0.69 6.49 5.01

± ± ± ±

0.19 0.08 0.39 0.42

7.57 ± 0.15 0.81 ± 0.24 1.06 ± 0.32

64

Cu-DOTA−NGR1

HT-29

64

Cu-DOTA−NGR2

percent injected dose/gram (% ID/g) 0.61 ± 0.11 0.58 ± 0.12 0.49 ± 0.09 0.65 ± 0.10 5.94 ± 0.33 5.25 ± 0.23 4.25 ± 0.38 4.57 ± 0.40 tumor-to-normal tissue uptake ratio 1.25 ± 0.16 0.91 ± 0.12 0.11 ± 0.08 0.13 ± 0.06 0.15 ± 0.09 0.14 ± 0.07

64

Cu-DOTA−NGR1 0.49 0.43 5.54 3.95

± ± ± ±

0.15 0.11 0.32 0.48

1.15 ± 0.18 0.09 ± 0.07 0.13 ± 0.12

64

Cu-DOTA−NGR2 0.65 0.40 4.95 5.24

± ± ± ±

0.21 0.12 0.45 0.39

1.62 ± 0.20 0.14 ± 0.08 0.12 ± 0.10

The results are presented as mean ± SD (n = 5/group).

NGR1 at the measured time points. Similar to the 64CuDOTA−NGR1 uptake in normal tissues, 64Cu-DOTA−NGR2 showed predominant uptake in liver and kidneys at the earlier time points (1, 2, and 4 h pi), and minimal uptake in most other organs (except for intestine) at all measured time points. As a function of time, the radioactivity was steadily cleared from the liver and rapidly excreted from the kidneys. Because the HT-1080 tumor uptake of 64Cu-DOTA−NGR2 was higher than that of 64Cu-DOTA−NGR1 at all measured time points, dimeric 64Cu-DOTA−NGR2 demonstrated overall better pharmacokinetics with higher contrast ratio of tumor-tonontarget as compared to monomeric 64Cu-DOTA−NGR1. The HT-1080 tumor and major organ uptake of 64Cu-DOTA− NGR1 and 64Cu-DOTA−NGR2 at 4 h pi are summarized in Table 1. For 64Cu-DOTA−NGR1, the ratio of HT-1080 tumor uptake to muscle, liver, and kidneys uptake at 4 pi was calculated to be 3.91 ± 0.19, 0.35 ± 0.21, and 0.52 ± 0.27, respectively; while the corresponding values for 64Cu-DOTA− NGR2 were 7.57 ± 0.15, 0.81 ± 0.24, and 1.06 ± 0.32, respectively. For CD13-negative HT-29 tumor model, 64CuDOTA−NGR1 and 64Cu-DOTA−NGR2 exhibited minimal tumor uptake and similar uptake in normal organs and tissues (liver, kidneys, and muscle) at 4 h pi as shown in Table 1. Blocking Experiment. The target specificity of 64CuDOTA−NGR1 and 64Cu-DOTA−NGR2 was achieved by a blocking experiment where the radiolabeled probe was coinjected with a cyclic NGR peptide (20 mg/kg of mouse body weight). Representative decay-corrected coronal slices that contained the tumor at 4 h pi of 64Cu-DOTA−NGR1 or 64 Cu-DOTA−NGR2 with coinjection of cyclic NGR peptide are illustrated in Figure 7. For 64Cu-DOTA−NGR1, the HT1080 tumor uptake in the presence of nonradiolabeled NGR peptide (0.61 ± 0.11% ID/g) was significantly lower than that without NGR peptide blocking (2.32 ± 0.17% ID/g) (P < 0.01) at 4 h pi (Table 1). The presence of nonradiolabled NGR peptide also slightly decreased the uptake of radiolabeled probe in liver and kidneys whereas the uptake of 64Cu-DOTA−NGR1 in muscle minimally altered between nonblocking and blocking group. As compared to 64Cu-DOTA−NGR1, 64Cu-DOTA− NGR2 demonstrated a similar pattern in terms of target specificity. The HT-1080 tumor uptake in the presence of nonradiolabeled NGR peptide (0.58 ± 0.12% ID/g) was significantly lower than that without NGR peptide blocking (5.22 ± 0.19% ID/g) (P < 0.01) at 4 h pi (Table 1). Taken together, the data confirmed that both 64Cu-DOTA−NGR1 and 64Cu-DOTA−NGR2 are CD13-specific PET probes.

Biodistribution Studies. The ex vivo biodistribution of Cu-DOTA−NGR1 and 64Cu-DOTA−NGR2 was examined in CD13-positive HT-1080 and CD13-negative HT-29 tumorbearing mice at 24 h pi. The percentage administered activity (injected dose) per gram of tissue (% ID/g) is shown in Figure 9. The biodistribution results were consistent with the 64

Figure 9. Biodistribution of 64Cu-DOTA−NGR1 or 64Cu-DOTA− NGR2 in HT-1080 or HT-29 tumor bearing athymic nude mice at 24 h pi (n = 5, mean ± SD).

quantitative analyses of microPET imaging. At 24 h pi, the HT-1080 tumor uptake of 64Cu-DOTA−NGR1 and 64CuDOTA−NGR2 reached 2.35 ± 0.17 and 3.61 ± 0.39% ID/g, respectively, whereas the HT-29 tumor uptake of 64CuDOTA−NGR1 [0.38 ± 0.09% ID/g (P < 0.01)] and 64CuDOTA−NGR2 [0.61 ± 0.09% ID/g (P < 0.01)] remained at a minimal level. In addition, both 64Cu-DOTA−NGR1 and 64CuDOTA−NGR2 displayed minimal uptake in most organs at 24 h pi except for high accumulation and retention in liver. For the HT-1080 tumor group, 4.14 ± 0.26% ID/g and 4.30 ± 0.36% ID/g remained in liver for 64Cu-DOTA−NGR1 and 64CuDOTA−NGR2, respectively. For the HT-29 tumor group, 3.64 ± 0.23% ID/g and 3.98 ± 0.26% ID/g remained in liver for 64 Cu-DOTA−NGR1 and 64Cu-DOTA−NGR2, respectively.

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DISCUSSION Positron emission tomography (PET) is a nuclear imaging technique used to map biological and physiological processes in living subjects after administration of radiolabeled probes.38,39 In oncology, [18F]fluorodeoxyglucose ([18F]FDG) is a most widely used PET probe. Although [18F]FDG proves to be quite useful for diagnosing tumor, identifying tumor grade and stage, and detecting recurrence in the clinic, [18F]FDG is not a target424

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specific PET probe.40,41 Over the past couple of decades, alternative PET probes to [18F]FDG that target specific biological processes in cancer biology have been extensively explored.15 Angiogenesis, the formation of new blood vessels from preexisting vasculature, is a fundamental process occurring during tumor progression.1,42,43 Research has shown that CD13 receptor is a critical regulator of angiogenesis. As a cell surface marker, CD13 receptor is overexpressed in many tumor cells.7 NGR-containing peptide has been identified as a specific ligand binding to CD13 receptor.22 Therefore, the development of NGR-containing derivatives for targeting CD13 in living subjects is of particular interest, which would enable valuable applications in the clinic for noninvasively detecting CD13 expression level, identifying patients who are appropriate candidates for treatment based on receptor expression/density, and accurately assessing the CD13-targeted therapy response. In addition to 18F and 11C, two conventional PET radionuclides used for the development of PET probes, several nonconventional metallic radionuclides, such as 64Cu, 68Ga, 86Y, and 89Zr, have been applied to PET probes.44 These metallic PET isotopes are usually characterized by longer half-lives, allowing the evaluation of radiopharmaceutical kinetics in the same subject to be achieved by successful PET imaging over a few hours or even days. Among these metallic radionuclides, 64 Cu (t1/2 = 12.7 h; β+ 655 keV, 17.8%) has attracted considerable interest because of its favorable decay half-life, low β+ energy, and commercial availability.45−47 In order to incorporate 64Cu into a biomolecule, a suitable chelator must be used. 64Cu will be captured by the corresponding chelator via coordination chemistry.47 The potential application of various chelating agents to the production of 64Cu-labeled PET probes has been previously summarized in the comprehensive reviews.45,48,49 Because of commercial availability of DOTA, we chose DOTA as a chelator to conjugate with NGR-containing peptides for 64Cu radiolabeling and employed the resulting PET probes for imaging CD13 receptor expression in vivo as a proof-of-principle study. Consequently, monomeric and dimeric NGR peptides were successfully synthesized and radiolabeled with 64Cu (Figures 1 and 2). The monomeric 64Cu-DOTA−NGR1 and dimeric 64 Cu-DOTA−NGR2 peptides were evaluated in vitro and in vivo for microPET imaging of CD13 receptor expression. The in vitro experiment demonstrated that both 64Cu-DOTA− NGR1 and 64Cu-DOTA−NGR2 are stable enough in PBS at room temperature and mouse serum at 37 °C for 24 h. More than 94% of 64Cu-DOTA−NGR1 or 64Cu-DOTA−NGR2 remained intact after 24 h of incubation in mouse serum at 37 °C (Figure 3). In order to select appropriate tumor cell lines for targeting CD13 receptor, we characterized the CD13 expression levels in human fibrosarcoma HT-1080 cells and human colon adenocarcinoma HT-29 cells. Both Western blot analysis and immunofluorescence staining confirmed that CD13 receptors are highly overexpressed in HT-1080 cells but not in HT-29 cells (Figure 4). Subsequently, the binding affinities of monomeric and dimeric NGR peptides to CD13 receptor were determined to be 1.27 ± 0.25 nM and 0.62 ± 0.29 nM, respectively (Figure 5), by using a HT-1080 cellbased competitive assay, indicating that NGR dimer had about 2-fold higher CD13 avidity than the corresponding monomeric NGR peptide presumably due to a bivalency effect. Cellular uptake study further revealed that both 64Cu-DOTA−NGR1 and 64Cu-DOTA−NGR2 bind to CD13-positive HT-1080

cells, but not to CD13-negative HT-29 cells (Figure 6). After 2 h incubation, the overall uptake of 64Cu-DOTA−NGR2 in HT1080 cells was higher than that of 64Cu-DOTA−NGR1, whereas uptake of both 64Cu-DOTA−NGR1 and 64CuDOTA−NGR2 remained minimal in CD13-negative HT-29 cells. Both 64Cu-DOTA−NGR1 and 64Cu-DOTA−NGR2 also displayed good retention in HT-1080 cells. After 2 h of cell efflux study, only about 0.4% of 64Cu-DOTA−NGR1 efflux and 0.70% of 64Cu-DOTA−NGR2 efflux were determined. These in vitro results justified further evaluations in animal models. MicroPET imaging and quantitative analysis of 64Cu-DOTA− NGR1 and 64Cu-DOTA−NGR2 in mice bearing HT-1080 tumor at 1, 2, 4, and 24 h after tail veil injection showed high HT-1080 tumor-to-background ratios. Accumulation of 64CuDOTA−NGR1 or 64Cu-DOTA−NGR2 was mainly in tumor, liver, kidneys, and intestine, while the probe uptake in most other organs was at a very low level. As a function of time, both 64 Cu-DOTA−NGR1 and 64Cu-DOTA−NGR2 were steadily cleared from the liver and rapidly excreted from the kidneys. At all measured time points, the HT-1080 tumor uptake of 64CuDOTA−NGR2 was higher than that of 64Cu-DOTA−NGR1, which might be attributed to the higher CD13 affinity of the NGR dimer. The increased molecular size of 64Cu-DOTA− NGR2 resulting in longer blood circulation time might be responsible for the prolonged tumor retention. Overall, dimeric 64 Cu-DOTA−NGR2 demonstrated better pharmacokinetics with higher contrast ratio of tumor-to-nontarget as compared to monomeric 64Cu-DOTA−NGR1 (Figures 7 and 8 and Table 1). The target specificity of 64Cu-DOTA−NGR1 and 64CuDOTA−NGR2 was achieved in CD13-negative mouse HT-29 xenografts, and a blocking experiment in mouse HT-1080 xenografts where the radiolabeled probe was coinjected with nonradiolabeled NGR peptide. In CD13-negative mouse HT29 xenografts, both 64Cu-DOTA−NGR1 and 64Cu-DOTA− NGR2 exhibited minimal tumor uptake (Table 1). In the blocking experiment of mouse HT-1080 xenografts, tumor uptake of both 64Cu-DOTA−NGR1 and 64Cu-DOTA−NGR2 was significantly reduced to a background level, suggesting that both 64Cu-DOTA−NGR1 and 64Cu-DOTA−NGR2 are CD13specific PET probes. The results from ex vivo biodistribution studies were consistent with the findings obtained from microPET imaging. For 64Cu labeling, DOTA has been reported as a commonly used chelator, and a series of 64Cu-DOTA peptide probes have been widely exploited for tumor imaging.50−52 However, high and prolonged liver uptake is problematic for 64Cu-DOTA probes, which was suggested to be the slow dissociation of 64Cu from the DOTA chelator.53 The instability of 64Cu-DOTA conjugates would result in demetalation and subsequent accumulation in nontarget tissues and organs such as in the liver. In this study, high accumulation and retention of 64CuDOTA−NGR1 and 64Cu-DOTA−NGR2 in mouse liver were also observed in both microPET imaging and biodistribution studies. Better chelation systems for 64Cu labeling such as TETA (1,4,8,11-tetraazacyclotetradecane-N,N′,N″,N‴-tetraacetic acid), cross-bridged cyclam ligands, and sarcophagine (3,6,10,13,16,19-hexaazabicyclo[6.6.6]icosane) may enhance the metal-chelate stability and subsequently lead to a better pharmacokinetic profile.54−56 Further improvements of 64Culabeled NGR peptide pharmacokinetics include the incorporation of an appropriate linker with suitable length, hydrophilicity, flexibility, and charges.31,57,58 Moreover, careful characterization of CD13 expression levels in human normal 425

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for antiangiogenic therapy. Semin. Oncol. 2001, 28 (5 Suppl. 16), 94− 104. (4) Kuwano, M.; Fukushi, J.; Okamoto, M.; Nishie, A.; Goto, H.; Ishibashi, T.; Ono, M. Angiogenesis factors. Intern. Med. 2001, 40 (7), 565−572. (5) Yancopoulos, G. D.; Davis, S.; Gale, N. W.; Rudge, J. S.; Wiegand, S. J.; Holash, J. Vascular-specific growth factors and blood vessel formation. Nature 2000, 407 (6801), 242−248. (6) Bhagwat, S. V.; Lahdenranta, J.; Giordano, R.; Arap, W.; Pasqualini, R.; Shapiro, L. H. CD13/APN is activated by angiogenic signals and is essential for capillary tube formation. Blood 2001, 97 (3), 652−659. (7) Guzman-Rojas, L.; Rangel, R.; Salameh, A.; Edwards, J. K.; Dondossola, E.; Kim, Y. G.; Saghatelian, A.; Giordano, R. J.; Kolonin, M. G.; Staquicini, F. I.; Koivunen, E.; Sidman, R. L.; Arap, W.; Pasqualini, R. Cooperative effects of aminopeptidase N (CD13) expressed by nonmalignant and cancer cells within the tumor microenvironment. Proc. Natl. Acad. Sci. U.S.A. 2012, 109 (5), 1637−1642. (8) Razak, K.; Newland, A. C. Induction of CD13 expression on fresh myeloid leukaemia: correlation of CD13 expression with aminopeptidase-N activity. Leuk. Res. 1992, 16 (6−7), 625−630. (9) Shipp, M. A.; Look, A. T. Hematopoietic differentiation antigens that are membrane-associated enzymes: cutting is the key! Blood 1993, 82 (4), 1052−1070. (10) Teranishi, J.; Ishiguro, H.; Hoshino, K.; Noguchi, K.; Kubota, Y.; Uemura, H. Evaluation of role of angiotensin III and aminopeptidases in prostate cancer cells. Prostate 2008, 68 (15), 1666−1673. (11) Ito, S.; Miyahara, R.; Takahashi, R.; Nagai, S.; Takenaka, K.; Wada, H.; Tanaka, F. Stromal aminopeptidase N expression: correlation with angiogenesis in non-small-cell lung cancer. Gen. Thorac. Cardiovasc. Surg. 2009, 57 (11), 591−598. (12) Terauchi, M.; Kajiyama, H.; Shibata, K.; Ino, K.; Nawa, A.; Mizutani, S.; Kikkawa, F. Inhibition of APN/CD13 leads to suppressed progressive potential in ovarian carcinoma cells. BMC Cancer 2007, 7, 140. (13) Massoud, T. F.; Gambhir, S. S. Molecular imaging in living subjects: seeing fundamental biological processes in a new light. Genes Dev. 2003, 17 (5), 545−580. (14) Ametamey, S. M.; Honer, M.; Schubiger, P. A. Molecular imaging with PET. Chem. Rev. 2008, 108 (5), 1501−1516. (15) Chen, K.; Chen, X. Positron emission tomography imaging of cancer biology: current status and future prospects. Semin. Oncol. 2011, 38 (1), 70−86. (16) Chen, K.; Conti, P. S. Target-specific delivery of peptide-based probes for PET imaging. Adv. Drug Delivery Rev. 2010, 62 (11), 1005− 1022. (17) Cai, J.; Liu, Z.; Wang, F.; Li, F. Phage display applications for molecular imaging. Curr. Pharm. Biotechnol. 2010, 11 (6), 603−609. (18) Deutscher, S. L. Phage display in molecular imaging and diagnosis of cancer. Chem. Rev. 2010, 110 (5), 3196−3211. (19) Arap, W.; Pasqualini, R.; Ruoslahti, E. Cancer treatment by targeted drug delivery to tumor vasculature in a mouse model. Science 1998, 279 (5349), 377−380. (20) Koivunen, E.; Gay, D. A.; Ruoslahti, E. Selection of peptides binding to the α5β1 integrin from phage display library. J. Biol. Chem. 1993, 268 (27), 20205−20210. (21) Koivunen, E.; Wang, B.; Ruoslahti, E. Isolation of a highly specific ligand for the α5β1 integrin from a phage display library. J. Cell Biol. 1994, 124 (3), 373−380. (22) Pasqualini, R.; Koivunen, E.; Kain, R.; Lahdenranta, J.; Sakamoto, M.; Stryhn, A.; Ashmun, R. A.; Shapiro, L. H.; Arap, W.; Ruoslahti, E. Aminopeptidase N is a receptor for tumor-homing peptides and a target for inhibiting angiogenesis. Cancer Res. 2000, 60 (3), 722−727. (23) von Wallbrunn, A.; Waldeck, J.; Holtke, C.; Zuhlsdorf, M.; Mesters, R.; Heindel, W.; Schafers, M.; Bremer, C. In vivo optical imaging of CD13/APN-expression in tumor xenografts. J. Biomed. Opt. 2008, 13 (1), 011007.

organs and tissues, such as in liver and kidneys, will warrant clinical imaging applications of NGR-containing PET probes for better somatic contrasts in particular.

■

CONCLUSION Monomeric and dimeric NGR peptides were successfully synthesized and radiolabeled with 64Cu for microPET imaging of CD13 receptor expression. As compared to the monomeric 64 Cu-DOTA−NGR1, the dimeric 64Cu-DOTA−NGR2 displayed better binding affinity and specificity with CD13-positive HT-1080 cells, and enhanced HT-1080 tumor uptake and improved HT-1080 tumor retention. The presumable bivalency effect and suitable size of 64Cu-DOTA−NGR2 make it a superior ligand for PET imaging of CD13 receptor expression in vivo. Given the fact that DOTA is a universal chelator capable of forming complexes with a variety of radiometals, such as 111 In, 67/68Ga, 64/67Cu, 86/90Y, and 177Lu, the same peptide conjugate used for 64Cu labeling in this study can also be applied to the development of other radiometal-labeled NGRcontaining probes for CD13-targeted tumor radioimaging and radiotherapy.

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AUTHOR INFORMATION

Corresponding Author

*K.C. and P.S.C.: tel, +1 (323) 442-3858; fax, +1 (323) 4423253; e-mail, [email protected] (K.C.), [email protected] (P.S.C.); University of Southern California, Department of Radiology, 2250 Alcazar Street, CSC103, Los Angeles, CA 90033 (K.C.). J.W.: tel, +86 029-84775449; fax, +86 02981230242; e-mail, [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the USC Department of Radiology, the Major Program of National Natural Science Foundation of China (Grant No. 81230033 and 81090274), the National Natural Science Foundation of China (Grant No. 30800275, 30970846, and 30970847), and the National Basic Research and Development Program of China (Grant No. 2011CB707704).

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ABBREVIATIONS USED APN, aminopeptidase N; NGR, Asn-Gly-Arg; PET, positron emission tomography; HPLC, high performance liquid chromatography; % ID/g, percentage injected dose per gram of tissue; pi, postinjection; PBS, phosphate buffered saline; DOTA, 1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid; Boc, t-butoxycarbonyl; NHS, N-hydroxysuccinimide; HATU, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo [4,5-b]-pyridinium 3-oxide hexafluorophosphate; HOAt, 1hydroxy-7-azabenzotriazole; TFA, trifluoroacetic acid; EDTA, ethylenediaminetetraacetic acid; DMSO, dimethyl sulfoxide; DMF, N,N-dimethylformamide; DIPEA, diisopropylethylamine

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REFERENCES

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dx.doi.org/10.1021/mp3005676 | Mol. Pharmaceutics 2013, 10, 417−427