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Jul 23, 2014 - CD13 receptor as a tumor vasculature biomarker has attracted great attention in cancer research. Through phage display screening, ...
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MicroPET Imaging of CD13 Expression Using a 64Cu-Labeled Dimeric NGR Peptide Based on Sarcophagine Cage Guoquan Li,†,‡ Xinlu Wang,†,§ Shu Zong,‡ Jing Wang,*,‡ Peter S. Conti,† and Kai Chen*,† †

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 710032, China § Department of Nuclear Medicine and PET-CT Center, Guangzhou General Hospital of Guangzhou Military Command, Guangzhou 510010, China S Supporting Information *

ABSTRACT: CD13 receptor as a tumor vasculature biomarker has attracted great attention in cancer research. Through phage display screening, NGR-containing peptides have been characterized as specific ligands binding to CD13 receptor. In this study, a 64 Cu-labeled dimeric NGR peptide based on sarcophagine cage was synthesized and evaluated for micropositron emission tomography (PET) imaging of CD13 expression in vivo. Macrocyclic chelating agent (sarcophagine cage) was conjugated with two azide moieties, followed by mixing with an alkyne-containing NGR peptide to rapidly provide the Sar-NGR2 peptide by click chemistry. Radiolabeling of Sar-NGR2 with 64Cu was achieved in >90% decay-corrected yield with radiochemical purity of >99%. The cell uptake study showed that 64Cu-Sar-NGR2 binds to CD13-positive HT-1080 cells, but not to CD13-negative MCF-7 cells. MicroPET imaging results revealed that 64Cu-Sar-NGR2 exhibits good tumor uptake in CD13-positive HT-1080 xenografts and significantly lower tumor uptake in CD13-negative MCF-7 xenografts. The CD13-specific binding of 64Cu-Sar-NGR2 was further verified by significant reduction of tumor uptake in HT-1080 tumor xenografts with coinjection of a nonradiolabeled NGR peptide. The biodistribution results demonstrated good tumor/muscle ratio (8.28 ± 0.37) of 64Cu-Sar-NGR2 at 24 h pi in HT-1080 tumor xenografts, which is in agreement with the quantitative analysis of microPET imaging. In conclusion, sarcophagine cage has been successfully applied to the construction of a 64Cu-labeled dimeric NGR-containing peptide. In vitro and in vivo studies demonstrated that 64Cu-Sar-NGR2 is a promising PET probe for imaging CD13 expression in living mice. KEYWORDS: PET imaging, NGR peptide, CD13, tumor angiogenesis, bivalency effect, 64Cu labeling, catalyst-free click chemistry

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INTRODUCTION

peptide demonstrated that the CNGRC loop is the key motif for tumor vasculature binding. Since then, numerous studies have been performed to reveal that CD13 receptor is the biological target of NGR-containing peptides.8 CD13 [also referred to aminopeptidase N (APN)] receptor is a zinc-dependent membrane-bound ectopeptidase, whose expression on activated blood vessels can be induced by angiogenic signals.9 Therefore, CD13 receptor is one of the critical regulators of angiogenesis.10 A sizable body of research showed that the high level of CD13 expression is associated with the progression of various tumors, such as prostate, lung, and ovarian tumor.11−14 Because of the important role of CD13

Molecular imaging with positron emission tomography (PET) creates the opportunity of noninvasive characterization of diseases.1 In order to acquire the PET imaging signals in a tested subject, molecular probes must be labeled with positronemitting radioisotopes prior to their administration.2 Numerous moieties, such as small molecules, peptides, protein, and nanoparticles, have been utilized as vehicles of PET probes.3 Among these functional moieties, peptides are of particular interest due to their intrinsic pharmacokinetic and tissue distribution patterns, high binding affinities, nonimmunogenic properties, and relatively easy production.3,4 The identification of unique peptides that bind to specific targets can be carried out through screening of bacteriophage (phage) display libraries.5,6 For example, a tumor vasculature homing phage carrying the sequence CNGRCVSGCAGRC was identified using in vivo phage display technique.7 Tumor homing of this sequence by the coinjection with a short cyclic CNGRC © 2014 American Chemical Society

Special Issue: Positron Emission Tomography: State of the Art Received: Revised: Accepted: Published: 3938

May 13, 2014 July 21, 2014 July 23, 2014 July 23, 2014 dx.doi.org/10.1021/mp500354x | Mol. Pharmaceutics 2014, 11, 3938−3946

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USA). MicroPET scans were performed on a microPET R4 scanner (Siemens Medical Solutions USA, Inc., Knoxville, TN). HPLC Methods. Analytical and semipreparative reversed phase HPLC for nonradiolabeled compounds were performed on a Thermo UltiMate 3000 HPLC system. Semipreparative reversed phase HPLC was carried out using a Phenomenex Luna C18 reversed phase column (5 μm, 250 × 10 mm). The flow rate was 4 mL/min, with the mobile phase starting from 95% solvent A (0.1% TFA in water) and 5% solvent B (0.1% TFA in acetonitrile) to 20% solvent A and 80% solvent B at 25 min. The UV absorbance was monitored at 214 and 254 nm. The analytical HPLC was performed using a Dionex Acclaim120 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 40% solvent A and 60% solvent B (0.1% TFA in acetonitrile) at 22.5 min. Purification of 64Cu-labeled peptide was achieved 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. A Phenomenex Luna C18 reversed phase analytical column (5 μm, 250 × 4.6 mm) was used, and the flow rate was 1 mL/min with the mobile phase starting from 100% solvent A (0.1% TFA in water) to 40% solvent A and 60% solvent B (0.1% TFA in acetonitrile) at 22.5 min. Synthesis of DBCO-NGR Peptide. To a solution of the monomeric NGR peptide (3.0 mg, 4.2 μmol) in 200 μL of sodium borate buffer (pH = 8.5) was added DBCO-PEG5-NHS ester32 (2.89 mg, 4.2 μmol) dissolved in 20 μL of DMSO. The mixture was adjusted to pH 8.5 and sonicated at room temperature for 1 h. The crude peptide was purified by HPLC. The desired product was collected and lyophilized to afford a white powder (4.7 mg, yield 86%). The retention time of DBCO-NGR peptide on analytical HPLC is 17.5 min. ESI-MS m/z C56H78N14O18S2 calcd, 1298.51; found, 1299.45 [M + H]+, 650.57 [M + 2H]2+. Synthesis of Diazido-Sar. To a solution of 4(azidomethyl)benzoic acid33 (6.2 mg, 35 μmol) in 100 μL of acetonitrile was added 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide, hydrochloric acid (EDC) (9.39 mg, 56 μmol) and N-hydroxysulfosuccinimide (sulfo-NHS) (10.6 mg, 49 μmol). After the mixture was adjusted to pH 5.5−6.0 using 0.1 M NaOH, the reaction was maintained at room temperature for 1 h. 1,8-Diamino-3,6,10,13,16,19-hexaazabicyclo[6.6.6]-eicosine (sarcophagine cage) (5.0 mg, 16 μmol) in 500 μL of sodium borate buffer (pH 8.5) was then added to the mixture. The reaction stayed at 4 °C overnight. The crude product was purified by HPLC. The desired product was collected and lyophilized to afford a white powder (3.85 mg, 38%). 1H NMR (CDCl3, 400 MHz), δ: 7.66 (d, J = 7.2 Hz, 4H), 7.43 (d, J = 7.2 Hz, 4H), 3.50−3.62 (m, 6H), 3.12−3.48 (m, 24H), 2.58 (s, 4H). ESI-MS m/z C30H44N14O2 calcd, 632.38; found, 633.48 [M + H]+, 317.38 [M + 2H]2+. Synthesis of Sar-NGR2 Peptide. To a solution of diazidoSar (0.95 mg, 1.5 μmol) in 50 μL of deionized water was added DBCO-NGR peptide (3.9 mg, 3.0 μmol) in 100 μL of deionized water. The mixture was incubated at room temperature for 1 h and purified by semipreparative HPLC. The desired peak was collected, concentrated, and lyophilized to afford the Sar-NGR peptide as a white powder (3.9 mg, 80%). The retention time of Sar-NGR2 peptide on analytical HPLC is 15.2 min. ESI-MS m/z C142H204N42O38S4 calcd,

receptor in tumor angiogenesis, noninvasive monitoring of CD13 expression levels in living subjects by using CD13specific molecular probes is of significance, which can facilitate the diagnosis of CD13 associated tumor angiogenesis and effectively monitor CD13-targeted antiangiogenic therapy. To date, various NGR peptides have been developed for CD13-targeted tumor imaging and/or therapy.15−20 Multimerization of a known ligand is one of the promising approaches to further enhance the binding affinity of the ligand to its presumed receptor in the development of imaging probes.21−28 Since CD13 receptor is located on the tumor cell surface, employment of NGR multimers for CD13-targeted imaging may provide an intriguing strategy to significantly enhance the “local concentration” of NGR ligands in the vicinity of CD13 receptor, leading to higher uptake and longer retention of NGR peptides in CD13-positive tumors. In our previous study,19 we capitalized on the bivalency principle and developed a dimeric NGR peptide, where glutamate was used as a branching linker to connect two monomeric NGR moieties. A macrocylic chelator, 1, 4, 7, 10-tetraazadodecaneN,N′,N″,N‴-tetraacetic acid (DOTA) was conjugated with the dimeric NGR peptide for 64Cu labeling. As compared to the monomeric NGR peptide, the dimeric NGR peptide displayed enhanced binding affinity to CD13-positive tumor cells and improved CD13-targeted tumor uptake. Our previous study verified that the construction of NGR multimers is a favorable approach for CD13-targeted imaging. Sarcophagine cage (denoted as “Sar”), a macrocyclic chelator, has been known for its excellent binding of Cu(II), thereby leading to high stability of its copper complex.29 On the basis of the chemical structure of sarcophagine cage, various bifunctional chelators have been recently developed.30,31 The resulting 64Cu-Sar complexes demonstrated high radiolabeling efficiency and good in vivo stability. To further explore the applications of Sar chelators, we constructed a new diazido-Sar chelator in this study. On the basis of diazido-Sar chelator, a new dimeric NGR peptide was successfully prepared using a catalyst-free click chemistry approach and radiolabeled with 64 Cu to afford 64Cu-Sar-NGR2. The in vitro properties of 64CuSar-NGR2 were subsequently investigated, including its stability, lipophilicity, binding affinity, and tumor cell uptake. The in vivo microPET imaging performance of 64Cu-Sar-NGR2 was assessed in subcutaneous CD13-positive HT-1080 fibrosarcoma and CD13-negetive MCF-7 breast adenocarcinoma mouse xenografts. CD13 specificity of 64Cu-Sar-NGR2 was further evaluated by in vivo blocking studies.

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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] was purchased from CS Bio Company, Inc. (Menlo Park, CA, USA). 1,8-Diamino-3,6,10,13,16,19-hexaazabicyclo[6.6.6]-eicosine (sarcophagine cage) was purchased from AREVA Med (Dallas, TX, USA). [64Cu]CuCl2 was obtained 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. 1H nuclear magnetic resonance (NMR) was recorded on a Varian 400 MHz spectrometer. Mass spectra were obtained on a ThermoElectron Finnigan LTQ mass spectrometer equipped with an electrospray ionization source (Thermo Scientific, 3939

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48-well plate at a density of 2.5 × 105 cells per well 24 h prior to the experiment. Tumor cells were then incubated with 64CuSar-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 with 0.25% trypsin/0.02% EDTA (Life Technologies, 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 (PerkinElmer 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, 64 Cu-Sar-NGR2 peptide (370 kBq/well) was first incubated with HT-1080 or MCF-7 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 with 0.25% trypsin/0.02% EDTA (Life Technologies, NY, USA). Cell suspensions were collected and measured in a gamma-counter (PerkinElmer 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 (Livermore, CA, USA). The HT-1080 human fibrosarcoma and MCF-7 human breast adenocarcinoma xenografts were generated by bilateral and subcutaneous injection of 5 × 106 tumor cells into the flanks 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-Sar-NGR2 peptide was intravenously injected into each mouse (n = 6/group) under isoflurane anesthesia. Five-minute static scans were acquired at 1, 2, and 4 h pi, and a 10 min static scan was acquired at 24 h pi. The images were reconstructed by a two-dimensional orderedsubsets expectation maximum (OSEM) algorithm. For each microPET scan, regions of interest (ROIs) were drawn over the tumor, normal tissue, and major organs on the decay-corrected whole-body coronal images. The radioactivity accumulation within the tumor, muscle, liver, and kidneys were obtained from the mean value within the multiple ROIs and then converted to %ID/g. For the blocking experiment, mice bearing HT-1080 and MCF-7 tumors (n = 6/group) were scanned at 1, 2, 4, and 24 h after the coinjection of 7.4 MBq of 64Cu-Sar-NGR2 with 20 mg/kg NGR peptide [c(CNGRC)] per mouse. Biodistribution Studies. The HT-1080 and MCF-7 tumor bearing nude mice (n = 6/group) were injected with 7.4 MBq of 64Cu-Sar-NGR2. At 24 h pi, 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 (PerkinElmer 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.

3233.42; found 1617.07 [M + 2H]2+, 1077.86 [M + 3H]3+, 808.88 [M + 4H]4+. 64 Cu Labeling and Formulation. [64Cu]Cu(OAc)2 was prepared by adding 37−111 MBq of [64Cu]CuCl2 in 0.1 M 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 Sar-NGR2 peptide (10 μg peptide per mCi 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-Sar-NGR2 peptide was collected and concentrated by rotary evaporation. The product was reconstituted in 500 μL of PBS and passed through a 0.22-μm Millipore filter into a sterile vial for use in the following experiments. Partition Coefficient. The partition coefficient value is expressed as log P, which was determined by measuring the distribution of 64Cu-Sar-NGR2 peptide in 1-octanol and PBS. Approximately 185 kBq of 64Cu-Sar-NGR2 peptide in 2 μL of PBS (pH = 7.4) was added to a vial containing 0.5 mL of 1octanol and 0.5 mL of PBS (pH = 7.4). After vortexing for 10 min, the vial was centrifuged at 12,500 rpm for 5 min. The octanol and PBS layer (200 μL of each layer) were pipetted into test tubes, respectively. The 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 64CuSar-NGR2 was tested in PBS and mouse serum. In brief, 3.7 MBq of 64Cu-Sar-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. For PBS study, an aliquot of the solution was directly taken at 1, 6, and 24 h after incubation, and the radiochemical purity was determined by reverse-phase HPLC under identical conditions. For mouse serum study, trifluoroacetic acid was added to the mixture at 1, 6, and 24 h after incubation, and the soluble fraction was filtrated 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 HT-1080 cell line and the human breast adenocarcinoma MCF-7 cell line were obtained from the American Type Culture Collection (Manassas, VA, USA). HT-1080 and MCF-7 cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2 in Dulbecco’s Modified Eagle Medium (DMEM) and supplemented with 10% fetal bovine serum (Life Technologies, NY, USA). 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 64Cu-labeled monomeric NGR peptide,19 in the presence of various concentrations of SarNGR2. 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 GraphPad 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.37,19 HT-1080 or MCF-7 cells were seeded into a 3940

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Figure 1. Schematic representation of the synthesis of 64Cu-Sar-NGR2. Reagents and conditions: (i) DBCO-PEG5-NHS, sodium borate buffer (pH 8.5); (ii) 4-(azidomethyl)benzoic acid, sulfo-NHS, EDC, sodium borate buffer (pH 8.5); (iii) deionized water, room temperature; (iv) 64CuCl2, 0.4 M NH4OAc (pH 5.5), 40 °C.

Figure 2. Stability of 64Cu-Sar-NGR2 in PBS (pH = 7.4) at room temperature and in mouse serum at 37 °C for 1, 6, and 24 h.

disulfide Cys:Cys = 4−8]. In the first step, coupling of the NHS ester activated DBCO-PEG5-COOH with monomeric NGR peptide afforded the DBCO-NGR peptide in a very good yield of 86%. The diazido-Sar was then constructed from 4(azidomethyl)benzoic acid and a commercially available Sar cage through amide coupling. In the third step, the catalyst-free click chemistry provided a rapid conjugation of DBCO-NGR peptide with the diazido-Sar in water. The desired Sar-NGR2 peptide was isolated through HPLC purification, and the purity was over 98%. The 64Cu-labeling (n = 6) was achieved in >85%

Statistical Analysis. Quantitative data were expressed as mean ± SD. Means were compared using one-way ANOVA and student’s t test. P values < 0.05 were considered statistically significant.

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RESULTS Chemistry and Radiochemistry. The schematic molecular structure of 64Cu-Sar-NGR2 peptide is shown in Figure 1. Synthesis of Sar-NGR2 peptide was accomplished in three steps starting from monomeric NGR peptide [GGGCNGRC; 3941

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decay-corrected yield for 64Cu-Sar-NGR2 with radiochemical purity of >99%. 64Cu-Sar-NGR2 peptide was purified and analyzed by HPLC. On the analytical HPLC, no significant difference of retention time between 64Cu-labeled probe and unlabeled conjugate was observed. The specific activity of 64CuSar-NGR2 was estimated to be about 37 MBq/nmol. The 64Culabeled probe was used immediately after formulation. Log P Value and in Vitro Stability. The octanol/water partition coefficient (log P) for 64Cu-Sar-NGR2 was determined to be −2.08 ± 0.15, suggesting that 64Cu-Sar-NGR2 is quite hydrophilic. The in vitro stability of 64Cu-Sar-NGR2 was studied in PBS (pH 7.4) at room temperature or mouse serum at physiological temperature 37 °C for 1, 6, and 24 h. The stability was presented as percentage of intact radiolabeled probe based on the HPLC analysis. As shown in Figure 2, 64CuSar-NGR2 exhibited excellent stability in PBS at room temperature as well as in mouse serum at 37 °C. After 24 h incubation, >96.0% of 64Cu-Sar-NGR2 in PBS at room temperature and >95.1% of 64Cu-Sar-NGR2 at 37 °C in mouse serum remained as the parent radiolabeled probe, indicating that 64Cu-Sar-NGR2 is stable in vitro. Cell-Based Binding Assay. The Western blot analysis and immunofluorescence staining of CD13 expression in HT-1080 and MCF-7 cells are shown in Figure S1 (Supporting Information). The results demonstrated that HT-1080 is a positive cell line for CD13 expression, while MCF-7 cell line is negative. HT-1080 cells were then used to measure the CD13 receptor binding affinity of Sar-NGR2 peptide by a competitive cell-binding assay, where a 64Cu-labeled monomeric NGR peptide was used as a CD13-specific radioligand for competitive displacement. The IC50 values of Sar-NGR2, which represent its concentration required to displace 50% of 64Cu-labeled monomeric NGR peptide bound to the HT-1080 cells, were determined to be 1.04 ± 0.26 nM (Figure 3). The cell-based binding assay showed that Sar-NGR2 has a similar CD13 avidity as compared to that of a dimeric NGR peptide, which was previously reported.19 Cell Uptake and Efflux. Cell uptake and retention of 64CuSar-NGR2 was examined in CD13-positive HT-1080 and CD13-negative MCF-7 tumor cells. The cell uptake study revealed that 64Cu-Sar-NGR2 binds to HT-1080 cells, but not

to MCF-7 cells. The 64Cu-Sar-NGR2 uptake in HT-1080 cells was determined to be about 0.72 ± 0.01% during the first hour of incubation. After 2 h of incubation, the uptake of 64Cu-SarNGR2 in HT-1080 cells significantly increased to 1.72 ± 0.24% of total input radioactivity (Figure 4 left, solid line). The cell efflux study demonstrated that 64Cu-Sar-NGR2 exhibits reasonable cell retention in HT-1080 cells. During 2 h of cell efflux study, only about 0.66% (from 1.72% to 1.06% of total input radioactivity) of 64Cu-Sar-NGR2 efflux was determined (Figure 4 right, solid line). In the CD13-negative MCF-7 cells, both of cellular uptake and retention of 64Cu-Sar-NGR2 were observed at a minimal level of total input radioactivity after 2 h incubation. The values were determined to be 0.4 ± 0.03% for MCF-7 cell uptake and 0.02 ± 0.002% for MCF-7 cell efflux, respectively (Figure 4, dotted line), which were significantly lower than those for HT-1080 cells (P < 0.05). MicroPET Imaging. The tumor-targeting efficacy and biodistribution pattern of 64Cu-Sar-NGR2 were evaluated in nude mice bilaterally bearing human fibrosarcoma HT-1080 and human breast adenocarcinoma MCF-7 xenografts (n = 6) at 1, 2, 4, and 24 h with static microPET scans. The CD13positive HT-1080 tumors were clearly visible with high contrast to contralateral background at all of time points measured after injection of 64Cu-Sar-NGR2; whereas the CD13-negative MCF7 tumors showed minimal uptake of 64Cu-Sar-NGR2. The representative decay-corrected coronal slice that contained the tumors at 4 h pi is shown in Figure 5 (left). Predominant uptake of 64Cu-Sar-NGR2 was also observed in the liver and kidneys at the early time points. Radioactivity accumulations in tumors and major organs for microPET scans were quantified by measuring the ROIs that encompassed the entire organ on the coronal images. The time−activity curves of HT-1080 tumor, MCF-7 tumor, liver, kidneys, and muscle after injection of 64Cu-Sar-NGR2 are depicted in Figure 6. The HT-1080 tumor uptake of 64Cu-Sar-NGR2 was calculated to be 6.83 ± 0.55, 5.99 ± 0.38, 5.42 ± 0.27, and 3.30 ± 0.33 %ID/g at 1, 2, 4, and 24 h pi, respectively. As a function of time, radioactivity was rapidly excreted from the kidneys and steadily cleared from the liver. The kidney uptake values were calculated to be 9.20 ± 0.56, 7.89 ± 0.38, 5.21 ± 0.37, and 1.42 ± 0.33 %ID/g at 1, 2, 4, and 24 h pi, respectively. The live uptake values were calculated to be 9.81 ± 0.62, 7.75 ± 0.66, 6.79 ± 0.39, and 4.20 ± 0.65 %ID/g at 1, 2, 4, and 24 h pi, respectively. Accumulation of 64Cu-Sar-NGR2 in most other organs (except for intestine) was at a very low level. The HT-1080 tumor, MCF-7 tumor, and major organ uptake of 64Cu-Sar-NGR2 at 4 h pi are summarized in Table 1. The ratio of HT-1080 tumor uptake to muscle, liver, and kidneys uptake at 4 pi was calculated to be 8.32 ± 0.18, 0.79 ± 0.27, and 1.04 ± 0.29, respectively. For CD13-negative MCF-7 tumors, 64Cu-Sar-NGR2 exhibited minimal tumor uptake at 4 h pi (Table 1). Blocking Experiment. The CD13 specificity of 64Cu-SarNGR2 was achieved by a blocking experiment where the radiolabeled probe was coinjected with a cyclic NGR peptide (20 mg/kg of mouse body weight). A representative decaycorrected coronal slice that contained the HT-1080 and MCF-7 tumors at 4 h pi of 64Cu-Sar-NGR2 with coinjection of a cyclic NGR peptide is shown in Figure 5 (right). The HT-1080 tumor uptake of 64Cu-Sar-NGR2 in the presence of a nonradiolabeled NGR peptide (1.05 ± 0.22 %ID/g) was significantly lower than that without NGR peptide blocking (5.42 ± 0.27 %ID/g) (P < 0.01) at 4 h pi (Table 1); whereas the uptake of 64Cu-Sar-NGR2 in other major organs (liver,

Figure 3. In vitro inhibition of Sar-NGR2 peptide bound to CD13 receptors on HT-1080 cells by a 64Cu-labeled monomeric NGR peptide. The IC50 value of Sar-NGR2 was calculated to be 1.04 ± 0.26 nM. 3942

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Figure 4. Cell uptake and efflux assay. Time-dependent cell uptake (left) and efflux (right) of 64Cu-Sar-NGR2 (n = 3, mean ± SD) using HT-1080 cells (solid line) and MCF-7 cells (dotted line).

Table 1. Decay-Corrected Biodistribution of 64Cu-Sar-NGR2 at 4 h Postinjection in Tumor-Bearing Mice Quantified by MicroPET Imaginga tissue tumor (T) muscle (M) liver (L) kidneys (K)

Figure 5. Representative decay-corrected coronal microPET images of mice bilaterally bearing HT-1080 and MCF-7 tumors (n = 6/group) after intravenous (i.v.) administration of 7.4 MBq of 64Cu-Sar-NGR2. The blocking study was performed by i.v. injection of 7.4 MBq of 64 Cu-Sar-NGR2 with coinjection of 20 mg/kg of NGR peptide [c(CNGRC)] as a blocking agent (n = 6/group). MicroPET images are shown at 4 h pi. Tumors are indicated by arrows.

T/M T/L T/K a

HT-1080

HT-1080 blockade

MCF-7 blockade

MCF-7

Percent Injected Dose/Gram (%ID/g) 5.42 ± 0.27 1.05 ± 0.22 0.75 ± 0.19 0.65 ± 0.09 0.58 ± 0.12 0.48 ± 0.13

0.56 ± 0.14 0.42 ± 0.17

6.79 ± 0.39 5.21 ± 0.37

6.15 ± 0.35 5.04 ± 0.41

5.93 ± 0.41 4.08 ± 0.36

Tumor-to-Normal Tissue Uptake Ratio 8.32 ± 0.18 1.81 ± 0.16 1.56 ± 0.15 0.79 ± 0.27 0.17 ± 0.03 0.12 ± 0.11 1.04 ± 0.29 0.20 ± 0.13 0.15 ± 0.10

1.33 ± 0.08 0.09 ± 0.11 0.14 ± 0.12

6.36 ± 0.23 5.35 ± 0.45

The results are presented as mean ± SD (n = 6/group).

administered activity (injected dose) per gram of tissue (% ID/g) is shown in Figure 7. The biodistribution results were in agreement with the quantitative analyses of microPET imaging. At 24 h pi, the HT-1080 tumor uptake of 64Cu-Sar-NGR2 reached 2.98 ± 0.60 %ID/g, whereas the MCF-7 tumor uptake of 64Cu-Sar-NGR2 [0.41 ± 0.29 %ID/g (P < 0.01)] remained at a minimal level. In addition, the HT-1080 tumor uptake of 64 Cu-Sar-NGR2 in the blocking group (0.71 ± 0.13 %ID/g) was significantly lower than that in the nonblocking group [2.98 ± 0.60 %ID/g (P < 0.01)]. Overall, 64Cu-Sar-NGR2 exhibited minimal uptake in most organs (85% (decay-corrected). To ascertain the utility of 64Cu-Sar-NGR2 as a PET probe for CD13 imaging, 64Cu-Sar-NGR2 was subjected to in vitro and in vivo testing. The stability experiment demonstrated that 64CuSar-NGR2 is quite stable in PBS at room temperature and mouse serum at 37 °C for 24 h. More than 95% of 64Cu-Sar-

NGR2 remained intact after 24 h incubation in mouse serum at 37 °C (Figure 2). In our previous studies, we selected the human fibrosarcoma HT-1080 as a CD13-positive cell line and the human colorectal adenocarcinoma HT-29 as a CD13negative cell line.19 In order to establish a bilateral tumor mouse model, we selected the human breast adenocarcinoma MCF-7 as a CD13-negative cell line in this study. Western blot analysis and immunofluorescence staining confirmed that CD13 receptors are highly overexpressed in HT-1080 cells but not in MCF-7 cells (Figure S1, Supporting Information). Using a cell-based competitive assay, the binding affinity of 64 Cu-Sar-NGR2 to CD13 receptor in HT-1080 cells was determined to be 1.04 ± 0.26 nM (Figure 3), which is similar to a dimeric NGR peptide that we previously reported.19 This result further demonstrated that multimerization of NGR peptide indeed enhances the CD13 binding affinity presumably due to a multivalency effect. Cellular uptake study revealed that 64 Cu-Sar-NGR2 binds to CD13-positive HT-1080 cells, but not to CD13-negative MCF-7 cells (Figure 4). After 2 h incubation, the uptake of 64 Cu-Sar-NGR2 in HT-1080 cells was significantly higher than that in CD13-negatice MCF-7 cells. 64 Cu-Sar-NGR2 also exhibited good retention in HT-1080 cells. The cell efflux study demonstrated that only about 0.66% of 64Cu-Sar-NGR2 efflux was determined after 2 h incubation. To better demonstrate the CD13 specificity of 64Cu-SarNGR2, we established an animal model with bilateral implantation of CD13-positive HT-1080 and CD13-negative MFC-7 tumors. MicroPET imaging of 64Cu-Sar-NGR2 in tumor-bearing mice at 1, 2, 4, and 24 h after tail veil injection showed high HT-1080 tumor-to-background ratio and minimal uptake in MCF-7 tumors. Accumulation of 64Cu-Sar-NGR2 was predominately in HT-1080 tumor, liver, and kidneys, while the 64 Cu-Sar-NGR2 uptake in other organs was at a very low level. As a function of time, 64Cu-Sar-NGR2 was rapidly excreted from the kidneys and steadily cleared from the liver. 64Cu-SarNGR2 also displayed good HT-1080 tumor retention. At 24 h pi, about 3.30 ± 0.33 %ID/g of 64Cu-Sar-NGR2 remained in 3944

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Molecular Pharmaceutics HT-1080 tumor, which may be due to high CD13 binding affinity and relatively large molecular size of 64Cu-Sar-NGR2. Overall, 64Cu-Sar-NGR2 displayed favorable pharmacokinetics with high tumor-to-normal tissue contrast (Figures 5 and 6 and Table 1). The CD13 specificity of 64Cu-Sar-NGR2 was achieved in CD13-negative MCF-7 tumors and a blocking experiment with coinjection of 64Cu-Sar-NGR2 and a nonradiolabeled NGR peptide. As shown in Figures 5 and 6 and Table 1, 64Cu-Sar-NGR2 displayed minimal MCF-7 tumor uptake. In the blocking experiment, HT-1080 tumor uptake of 64 Cu-Sar-NGR2 was significantly reduced to a background level, indicating that 64Cu-Sar-NGR2 is a CD13-specific PET probe. The results from ex vivo biodistribution studies were in agreement with the microPET imaging data (Figure 7). Despite of excellent in vivo CD13 specificity of 64Cu-Sar-NGR2, we observed high radioactivity accumulation in mouse liver, which is similar to what we detected in our previous study of 64CuDOTA-NGR2. This observation may be caused by a certain amount of CD13 expression in mouse liver and trapping of 64 Cu in mouse liver tissue. Our ongoing efforts are focusing on addressing these issues to warrant translational studies of 64CuSar-NGR2 for PET imaging of CD13 expression.

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CONCLUSIONS

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ASSOCIATED CONTENT

ABBREVIATIONS

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REFERENCES

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; Sar, 1,8-diamino-3,6,10,13,16,19-hexaazabicyclo[6.6.6]eicosine; sulfo-NHS, N-hydroxysulfosuccinimide; EDC, 1ethyl-3-(3-(dimethylamino)propyl)-carbodiimide; DBCO, dibenzocyclooctyne

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A dimeric NGR-containing peptide based on a sarcophagine cage (64Cu-Sar-NGR2) was successfully synthesized using a catalyst-free click chemistry approach and radiolabeled with 64 Cu for microPET imaging of CD13 expression. 64Cu-SarNGR2 displayed good binding affinity and CD13 specificity with HT-1080 cells and excellent HT-1080 tumor uptake. The bivalency effect and suitable size of 64Cu-Sar-NGR2 make it an excellent PET probe. Considering that sarcophagine cage is also capable of forming complexes with other radiometals, such as 67/68 Ga, the approach used in this study may be applied to the development of other radiometal-labeled peptide-based probes for tumor diagnosis and treatment.

S Supporting Information *

Western blot analysis and immunofluorescence staining. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*(K.C.) Tel: +1 (323) 442-3858. Fax: +1 (323) 442-3253. Email: [email protected]. *(J.W.) Tel: +86 029-84775449. Fax: +86 029-81230242. Email: [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). 3945

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