Comparison of Biological Properties of ^99mTc-Labeled Cyclic RGD Peptide Trimer and Dimer Useful as SPECT Radiotracers for Tumor Imaging

Abstract

Introduction

This study sought to evaluate a ^99mTc-labeled trimeric cyclic RGD peptide (^99mTc-4P-RGD₃) as the new radiotracer for tumor imaging. The objective was to compare its biological properties with those of ^99mTc-3P-RGD₂ in the same animal model.

Methods

HYNIC-4P-RGD₃ was prepared by reacting 4P-RGD₃ with excess HYNIC-OSu in the presence of diisopropylethylamine. ^99mTc-4P-RGD₃ was prepared using a kit formulation, and evaluated for its tumor-targeting capability and biodistribution properties in the BALB/c nude mice with U87MG human glioma xenografts. Planar and SPECT imaging studies were performed in athymic nude mice with U87MG glioma xenografts. For comparison purpose, ^99mTc-3P-RGD₂ (a α_vβ₃-targeted radiotracer currently under clinical evaluation for tumor imaging in cancer patients) was also evaluated in the same animal models. Blocking experiments were used to demonstrate the α_vβ₃ specificity of ^99mTc-4P-RGD₃.

Results

^99mTc-4P-RGD₃ was prepared with >95% RCP and high specific activity (~200 GBq/µmol). ^99mTc-4P-RGD₃ and ^99mTc-3P-RGD₂ shared almost identical tumor uptake and similar biodistribution properties. ^99mTc-4P-RGD₃ had higher uptake than ^99mTc-3P-RGD₂ in the intestines and kidneys; but it showed better metabolic stability. The U87MG tumors were clearly visualized by SPECT with excellent contrast with ^99mTc-4P-RGD₃ and ^99mTc-3P-RGD₂.

Conclusion

Increasing peptide multiplicity from 3P-RGD₂ to 4P-RGD₃ offers no advantages with respect to the tumor-targeting capability. ^99mTc-4P-RGD₃ is as good a SPECT radiotracer as ^99mTc-3P-RGD₂ for imaging α_vβ₃-positive tumors.

INTRODUCTION

Integrin α_vβ₃ is a receptor for the extracellular matrix proteins (e.g. collagen, fibrinogen, fibronectin, laminin and vitronectin) with one or more arginine-glycine-aspartic (RGD) tripeptide sequences. The α_vβ₃ is generally expressed at low levels on epithelial cells and mature endothelial cells, but it is overexpressed on the tumor cells and activated endothelial cells of neovasculature. Because of the role of α_vβ₃ in tumor angiogenesis and metastasis, cyclic RGD peptides are often used as α_vβ₃ antagonists for tumor therapy, and radiolabeled cyclic RGD peptides are utilized as “α_vβ₃–targeted” radiotracers for tumor imaging [1–10]. Over the last several years, we have been interested in radiolabeled multimeric cyclic RGD peptides as radiotracers for imaging α_vβ₃-positive tumors and related metastasis [11–29]. Multiple cyclic RGD moieties are utilized to maximize their α_vβ₃ binding affinity and tumor uptake of their corresponding radiotracers regardless of the attached isotope. We found that there are two important factors (bivalency and locally enhanced RGD concentration) contributing to the higher α_vβ₃ binding affinity of multimeric cyclic RGD peptides than their monomeric analogs [1,20,23]. The concentration factor exists in all multimeric RGD peptides regardless of the linker length. The key to achieve bivalency is the distance between two cyclic RGD motifs. We also found that cyclic RGD tetramers (such as RGD₄) are actually bivalent in binding to the α_vβ₃ even though they have four identical cyclic c(RGDfK) motifs [20,23]. Among many of radiolabeled multimeric cyclic peptides, ^99mTc-3P-RGD₂ (Figure 1) and ¹⁸F-Alfatide-II are currently under clinical evaluations as new radiotracers for tumor imaging in cancer patients [30–36]. Since ^99mTc-3P-RGD₂ could be prepared in >95% radiochemical purity, it offers significant advantages over ¹⁸F-Alfatide-II, which often requires post-labeling chromatographic purification [35,36]. ^99mTc-3P-RGD₂ SPECT/CT has been used to quantify the tumor uptake [37–40], and to monitor the tumor growth [37,38], the progression of breast cancer lung metastases [39], and the pharmacological effects of antiangiogenic therapy [38,40].

Figure 1.

Chemdraw structures of multimeric cyclic RGD peptides (3P-RGD₂ and 4P-RGD₃) and their ternary ligand complexes [^99mTc(HYNIC-BM)(tricine)(TPPTS)] (BM = biomolecule; ^99mTc-3P-RGD₂: BM = 3P-RGD₂; and ^99mTc-4P-RGD₃: BM = 4P-RGD₃). ^99mTc-4P-RGD₃ is the new SPECT radiotracer evaluated in this study. ^99mTc-3P-RGD₂ is currently under intensive clinical evaluation for tumor imaging in cancer patients [31–35]. It was used for comparison purposes to study the impact of peptide multiplicity on tumor uptake and biodistribution properties in the same tumor-bearing animal model.

In the literature, only a few radiolabeled cyclic RGD trimers were reported [41,42]. There was no comparison in biodistribution properties between the radiolabeled cyclic RGD dimers and trimers. With this in mind, we designed a new RGD peptide trimer (Figure 1: 4P-RGD₃ = PEG₄-ACHDA[cyclo[Arg-Gly-Asp-D-Phe-Lys(PEG₄)]]₃; and ACHDA = 4-amino-4-(2-carboxyethyl)heptanedioic acid). ACHDA was used to bridge three c(RGDfK) moieties. Four PEG₄ linkers were used to enhance the hydrophilicity of ^99mTc radiotracer and increase the distance between two neighboring c(RGDfK) moieties in 4P-RGD₃ so that it is able to achieve bivalency. In this report, we present the synthesis and biological evaluation of ^99mTc-4P-RGD₃ (Figure 1) as a new SPECT radiotracer for tumor imaging. ^99mTc-4P-RGD₃ is the first ^99mTc-labeled cyclic RGD peptide trimer. The main objective of this study was to compare the biodistribution and imaging properties of ^99mTc-4P-RGD₃ and ^99mTc-3P-RGD₂ in the same tumor-bearing animal model.

EXPERIMENTAL SECTION

Materials and Instruments

Tricine and trisodium triphenylphosphine-3,3',3"-trisulfonate (TPPTS) were purchased from Sigma/Aldrich (St. Louis, MO), and were used without further purification. Cyclic RGD peptides RGD₂ (E[c(RGDfK)]₂ = Glu[cyclo(Arg-Gly-Asp-D-Phe-Lys)]₂) and 4P-RGD₃ (PEG₄-ACHDA{cyclo[Arg-Gly-Asp-D-Phe-Lys(PEG₄)]}₃) were custom-made by the Peptides International, Inc. (Louisville, KY). Sodium succinimidyl 6-(2-(2-sulfonatobenzaldehyde)hydrazono)nicotinate (HYNIC-OSu) was prepared according to literature method [43]. HYNIC-3P-RGD₂ and ^99mTc-3P-RGD₂ were prepared using the procedure described in our previous report [14]. Na^99mTcO₄ was obtained from Cardinal HealthCare® (Indianapolis, IN). The MALDI (matrix-assisted laser desorption ionization) mass spectral data for HYNIC-4P-RGD₃ were collected on an Applied Biosystems Voyager DE PRO mass spectrometer (Framingham, MA), the Department of Chemistry, Purdue University.

HPLC Methods

HPLC Method 1 used a LabAlliance HPLC system (Scientific Systems, Inc., State College, PA) equipped with a UV/vis detector (λ=254 nm) and Zorbax C₁₈ column (9.4 mm × 250 mm, 100 Å pore size; Agilent Technologies, Santa Clara, CA). The flow rate was 2.5 mL/min with a mobile phase being 90% A and 10% B at 0 min to 80% A and 20% B at 5 min, and to 50% A and 50% B at 20 min. The radio-HPLC (Method 2) used an Agilent HP-1100 HPLC system (Agilent Technologies, Santa Clara, CA) equipped with a β-ram IN/US detector (Tampa, FL) and Zorbax C₈ column (4.6 mm × 250 mm, 300 Å pore size; Agilent Technologies, Santa Clara, CA). The flow rate was 1 mL/min. The mobile phase was isocratic for the first 5 min with 90% A (25 mM NH₄OAc, pH = 6.8) and 10% B (acetonitrile), followed by a gradient mobile phase going from 90% A and 10% B at 5 min to 40% A and 60% B at 20 min. The radiochemical purity was reported as the percentage of area for the peak at 15 – 16 min on each radio-HPLC chromatogram of ^99mTc-3P-RGD₂ and ^99mTc-4P-RGD₃. The instant thin layer chromatography (ITLC) used Gelman Sciences silica-gel strips and a 1:1 mixture of acetone and saline as the mobile phase. ^99mTc-3P-RGD₂, ^99mTc-4P-RGD₃ and ^99mTcO₄⁻ migrated to solvent front while [^99mTc]colloid stayed at the origin. [^99mTc]colloid was reported as the percentage of radioactivity at the origin over the total radioactivity on each strip.

HYNIC-4P-RGD₃

HYNIC-OSu (13.5 mg, 30 µmol) and 4P-RGD₃ (9.0 mg, 3 µmol) were dissolved in DMF (2 mL). After addition of excess DIEA (5 drops), the mixture was stirred at room temperature for 24 h. To the mixture was added 2 mL of water after completion of the reaction. The pH value was then adjusted to 3 – 4 using neat TFA. The product was separated from the reaction mixture by HPLC. Fractions at ~18 min were collected. Lyophilization of collected fractions afforded the expected product HYNIC-4P-RGD₃ as a white powder. The yield was 6.5 mg (~50%). MALDI-MS: m/z = 3295.8240 for [M + H]⁺ (M = 3294.61 calcd. for [C₁₄₈H₂₂₇N₃₅O₄₈S]).

^99mTc-Labeling

To a lyophilized vial containing 20 – 25 µg HYNIC-RGD conjugate, 5 mg TPPTS, 6.5 mg tricine, 40 mg mannitol, 38.5 mg disodium succinate hexahydrate and 12.7 mg succinic acid was added 1.0 – 1.5 mL of Na^99mTcO₄ solution (1,110 – 1,850 MBq in saline). The reconstituted vial was heated at 100 °C for 20 min. After cooling at room temperature for ~5 min, a sample of the resulting solution was analyzed by radio-HPLC (Method 2). The radiochemical purity (RCP) was always >95% for imaging and biodistribution studies. The solution stability was monitored by HPLC for 6 h.

Dose Preparation

For biodistribution, doses were prepared by dissolving ^99mTc radiotracer in saline to a concentration of ~1 MBq/mL. For SPECT/CT imaging studies, doses were prepared by dissolving ^99mTc radiotracer in saline to ~370 MBq/mL. In the blocking experiments, RGD₂ was dissolved in the dose solution to 3.5 mg/mL. The resulting dose solution was filtered with a 0.20 µm syringe-driven Millex-LG filter before being injected into animals. Each animal was injected with ~0.1 mL of the dose solution.

Cell Culture and Animal Models for Biodistribution

All animal experiments were performed in accordance with guidelines of Institutional Animal Care and Use Committee (IACUC) at Fu-Wai Hospital (Beijing, China). U87MG cell line was obtained from Peking Union Medical College. U87MG cells were cultured in DMEM (Dulbecco's modified eagle medium nonessential amino acids sodium pyruvate), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin solution at 37 °C in a humidified atmosphere of 5% CO₂ in air, grown as monolayers, and were harvested or split when they reached 95% confluence to maintain exponential growth. Female BALB/c nude mice (4 – 5 weeks of age) were purchased from Department of Experimental Animals, Fu-Wai Hospital. The animal model was established by inoculating subcutaneously with 5 × 10⁶ of U87MG cells into the shoulder flank of each animal. When the tumor volume reached ~400 mm³ (~10 days after inoculation of U87MG cells), the tumor-bearing animals were used for biodistribution studies.

Biodistribution

Each tumor-bearing animal (16 – 20 g) was administered with ~0.1 MBq of ^99mTc radiotracer (~2 ng HYNIC-3P-RGD₂ or HYNIC-4P-RGD₃) by tail vein injection. Five animals were sacrificed by sodium pentobarbital overdose (~200 mg/kg) at 5, 30, 60 and 120 min p.i. Blood was withdrawn from the heart. Tumors and normal organs (brain, eyes, heart, intestines, kidneys, liver, lungs, muscle and spleen) were harvested, washed with saline, dried with absorbent tissue, weighed, and counted on a Perkin Elmer Wizard – 1480 γ-counter (Shelton, CT). The organ uptake was calculated as the percentage of injected dose per gram of tissue (%ID/g). The blocking experiment was performed using RGD₂, a well-known integrin α_vβ₃ antagonist [1,3,7], as the blocking agent. Each animal was administered with ~0.1 MBq of ^99mTc-4P-RGD₃ along with ~350 µg RGD₂ (~14 mg/kg). Biodistribution data (%ID/g) and tumor-to-background (T/B) ratios were expressed as the average plus standard deviation. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by the Newman-Keuls test for multiple comparisons. The level of significance was set at p < 0.05.

Cell Culture and Animal Model for Imaging Studies

Imaging studies were performed in compliance with the NIH animal experimentation guidelines (Principles of Laboratory Animal Care, NIH Publication No. 86-23, revised 1985). The protocol was approved by the Purdue University Animal Care and Use Committee (PACUC). U87MG, MDA-MB-435 and PC-3 cell lines were obtained from ATCC (Manassas, VA). U87MG cells were cultured in the Minimum Essential Medium, Eagle with Earle’s Balanced Salt Solution (non-essential amino acids sodium pyruvate). PC-3 cells were cultured in the F-12 medium (GIBCO, Grand Island, NY). MDA-MB-435 cells were grown in the RPMI Medium 1640 with L-glutamine (GIBCO, Grand Island, NY). All tumor cell lines were supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin solution, and grown at 37 °C in a humidified atmosphere of 5% CO₂ in air. Cells were grown as monolayers and were harvested or split when they reached 90% confluence. Female athymic nu/nu mice were purchased from Harlan at 4 – 5 weeks of age, and were implanted with 5 × 10⁶ tumor cells in 0.1 mL of saline into the shoulder flanks of each animal for the U87MG and PC-3 models, or breast fat pads for the MDA-MB-435 model. When the tumor size was 0.1 – 0.5 g, and animals were used for imaging studies.

Planar Imaging

Each animal was administered with 300~500 µCi of ^99mTc radiotracer in 0.1 mL saline via tail vein injection, anesthetized with intraperitoneal injection of ketamine (80 mg/kg) and xylazine (19 mg/kg), and then placed supine on a single head mini gamma camera (Diagnostic Services Inc., NJ) equipped with a parallel-hole, medium-energy and high-resolution collimator. The whole-body anterior images were acquired at 60 min p.i., and stored digitally in a 128 × 128 matrix. The acquisition count limits were set at 300 K. After completion of imaging, animals were sacrificed by sodium pentobarbital overdose (~200 mg/kg).

SPECT/CT Imaging

SPECT/CT images were obtained using a u-SPECT-II/CT scanner (Milabs, Utrecht, The Netherlands) equipped with a 1.0 mm multi-pinhole collimator. The tumor-bearing mouse was injected with ~37 MBq of ^99mTc radiotracer (~ 1.0 µg HYNIC-conjugated cyclic RGD peptide) in 0.1 mL saline via the tail vein. At 60 min p.i., the animal was placed into a shielded chamber connected to an isoflurane anesthesia unit (Univentor, Zejtun, Malta). Anesthesia was induced using an air flow rate of 350 mL/min and ~3.0% isoflurane. After induction of anesthesia, the animal was immediately placed supine on the scanning bed. The air flow rate was then reduced to ~250 mL/min with ~2.0% isoflurane. Rectangular scan in the regions of interest (ROIs) from SPECT and CT were selected on the basis of orthogonal optical images provided by the integrated webcams. After SPECT acquisition (75 projections over 30 min per frame, 2 frames), the animal was then transferred into the attached CT scanner and imaged using the ‘normal’ acquisition settings (2 degree intervals) at 45 kV and 500 µA. After CT acquisition, the animal was allowed to recover in a lead-shielded cage.

Image Data Processing

SPECT reconstruction was performed using a POSEM (pixelated ordered subsets by expectation maximization) algorithm with 6 iterations and 16 subsets. CT data were reconstructed using a cone-beam filtered back-projection algorithm (NRecon v1.6.3, Skyscan). After reconstruction, the SPECT and CT data were automatically co-registered according to the movement of the robotic stage, and then re-sampled to equivalent voxel sizes. Co-registered images were further rendered and visualized using the PMOD software (PMOD Technologies, Zurich, Switzerland). A 3D-Guassian filter (0.8 mm FWHM) was applied to smooth noise, and the LUTs (look up tables) were adjusted for good visual contrast. The reconstructed images were visualized as both orthogonal slices and maximum intensity projections.

Radioactivity Quantification

Radiation sources of known amount of radioactivity were imaged and reconstructed using the same scanning protocol described above. A standard curve was generated to correlate the pixel intensities in the reconstructed images to the radioactivity as measured by a γ-counter. Tumor delineation was performed on CT and SPECT images according to the literature method [37–40]. Briefly, the ROIs (regions of interest) were drawn manually to cover the entire tumor based on transverse view of the CT image. For the tumor delineation with SPECT, a threshold of 50% or more of the maximum pixel value on the SPECT image was chosen. In order to accurately represent the entire tumor, SPECT/CT was also employed to delineate the tumor ROIs. The tumor volume and radioactivity counts were generated by using the PMOD software (PMOD Technologies, Zurich, Switzerland). The amount of radioactivity in each tumor was calculated according to the above mentioned standard curve. The tumor uptake of ^99mTc radiotracer was expressed the percentage of injected dose per gram (%ID/g, assuming the tumor density is ~1 g/cm³), and was compared with the data from biodistribution in the same tumor-bearing animal model.

Metabolism

Athymic nude mice (n = 3) were used for metabolism study. Each animal was administered with ~10 MBq of ^99mTc-4P-RGD₃ via the tail vein. The urine samples were collected at 30 min and 120 min p.i. by manual void, counted for total radioactivity, and were mixed with equal volume of 50% acetonitrile aqueous solution. The mixture was centrifuged at 8,000 rpm. The supernatant was collected, counted for the recovered radioactivity, and filtered with a 0.20 µm Millex-LG filter to remove foreign particles. The filtrate was analyzed by HPLC (Method 2). The feces samples were collected at 120 min p.i., counted for total radioactivity, and suspended in 20% acetonitrile aqueous solution. The mixture was vortexed for ~5 min. After centrifuging at 8,000 rpm, the supernatant was collected, counted for the recovered radioactivity, and filtered with a 0.20 µm syringe-driving Millex-LG filter unit to remove foreign particles. The filtrate was then analyzed by radio-HPLC (Method 2). The percentage of radioactivity recovery was >95% by γ-counting for both urine and feces samples.

Tissue Immunohistochemistry

Tumor tissues were harvested, immediately snap-frozen in OCT (optical cutting temperature) solution and cut into 5 µm-thick slices. After thorough drying at room temperature, the slides were fixed with ice-cold acetone for 10 min. The sections were then blocked and incubated with anti-integrin β₃ antibody (1:100, Santa Cruz) and CD31 antibody (1:100, BD Biosciences) for 1 h at room temperature. After washing, DyLight 594 and fluorescein isothiocyanate (FITC)-conjugated second antibodies (1:500, Jackson ImmunoResearch Inc., West Grove, PA) were added to link to integrin β₃ or CD31 primary antibody, respectively. The fluorescence was visualized with an Olympus BX51 fluorescence microscope (Olympus America Inc., Center Valley, PA). Phase contrast pictures were also obtained as the histological reference to illustrate the texture of the tumor or normal organ tissue and demonstrate the site of fluorescent staining. All the pictures were taken under 100x magnification. The same procedure was used to compare integrin β₃ expression levels in the U87MG tumor, kidneys, liver and lungs.

RESULTS

Synthesis of HYNIC-4P-RGD₃

HYNIC-4P-RGD₃ was prepared from the reaction of 4P-RGD₃ with HYNIC-OSu in the presence of excess DIEA. The conjugation reaction was fast, and took ~24 h to complete at room temperature. HYNIC-4P-RGD₃ was purified by HPLC and characterized by ESI-MS. ESI-MS data was consistent with the proposed composition for HYNIC-4P-RGD₃. Its HPLC purity was > 95% before being used for ^99mTc-labeling.

Synthesis of ^99mTc-4P-RGD₃

^99mTc-4P-RGD₃ was prepared from the reaction of HYNIC-4P-RGD₃ with ^99mTcO₄⁻ in the presence of tricine and TPPTS coligands. ^99mTc-labeling was accomplished by heating the reconstituted vial at 100 °C for 10 – 15 min. Figure 2 shows radio-HPLC chromatograms of ^99mTc-4P-RGD₃ in the kit matrix at 1 h and 6 h post-labeling. Its RCP was >95% without post-labeling purification. The specific activity was high (~200 GBq/µmol). ^99mTc-4P-RGD₃ remained stable in the kit matrix for >6 h post-labeling.

Figure 2.

Radio-HPLC chromatograms of ^99mTc-4P-RGD₃ in the kit matrix at 1 h (top) and 6 h (bottom) post-labeling. The radiometric peak at ~15.6 min was from ^99mTc-4P-RGD₃. Its radiochemical purity was always >95% without post-labeling chromatographic purification.

Biodistribution Properties

The selected biodistribution data for ^99mTc-4P-RGD₃ are summarized in Table 1. ^99mTc-3P-RGD₂ was evaluated in the same animal model, and its selected biodistribution data are listed in Table 2. In general, ^99mTc-4P-RGD₃ and ^99mTc-3P-RGD₂ shared very similar biodistribution characteristics (Table 1 and 2). The tumor uptake of ^99mTc-4P-RGD₃ was almost identical to that of ^99mTc-3P-RGD₂ over the 2 h study period (Figure 3A). Increasing peptide multiplicity did not improve the radiotracer tumor uptake. In addition, they shared similar tumor/blood ratios (Figure 3B). The most significant difference between ^99mTc-4P-RGD₃ and ^99mTc-3P-RGD₂ is their uptake values in intestines (Figure 3C) and kidneys (Figure 3D). It is important to note that the uptake of ^99mTc-4P-RGD₃ and ^99mTc-3P-RGD₂ in the U87MG glioma xenografted in BALB/c nude mice was significantly lower than that reported for ^99mTc-3P-RGD₂ in previous studies [14,21,27]. To understand this difference, we performed immunohistochemical (IHC) staining on the tumor tissues obtained from U87MG-bearing BALB/c and athymic nude mice. We found that the U87MG tumor tissues obtained from the athymic nude mice had higher blood vessel density than that of the U87MG tissues from the BALB/c nude mice (Figure 3E). Obviously, the host animal species (e.g. BALB/c versus athymic nude mice) have significant impact on the radiotracer tumor uptake even if the same cancer cell line is used.

Table 1.

Selected biodistribution data and tumor-to-background ratios of ^99mTc-4P-RGD₃ in BALB/c nude mice (n = 5) bearing U87MG human glioma xenografts. The tumor uptake (%ID/g) is reported as an average plus/minus the standard deviation.

Organ	5 min	30 min	60 min	120 min
Blood	4.43 ± 0.15	1.44 ± 0.18	0.88 ± 0.13	0.46 ± 0.04
Brain	0.51 ± 0.04	0.53 ± 0.23	0.28 ± 0.05	0.21 ± 0.03
Eyes	3.36 ± 0.22	3.20 ± 0.18	3.93 ± 0.78	2.77 ± 0.46
Heart	6.11 ± 0.31	4.07 ± 0.46	4.11 ± 0.31	2.51 ± 0.46
Intestine	19.06 ± 1.23	14.31 ± 1.43	15.93 ± 3.26	11.68 ± 1.22
Kidneys	55.62 ± 3.10	46.99 ± 4.18	45.81 ± 4.35	35.59 ± 8.55
Liver	9.89 ± 0.35	6.76 ± 1.03	6.11 ± 0.91	5.58 ±0.97
Lungs	5.63 ± 3.66	4.93 ± 0.57	4.88 ± 0.43	2.64 ± 0.53
Muscle	3.05 ± 0.50	2.33 ± 0.37	3.28 ± 1.15	1.36 ± 0.16
Spleen	5.94 ± 0.55	6.15 ± 0.78	4.89 ± 1.10	5.13 ± 0.93
Tumor	5.42 ± 1.48	5.22 ± 0.17	5.17 ± 1.01	3.74 ± 0.51
Tumor/Blood	1.23 ± 0.34	3.69 ± 0.48	6.21 ± 2.34	8.21 ± 1.71
Tumor/Liver	0.55 ± 0.16	0.79 ± 0.11	0.87 ± 0.25	0.70 ± 0.19
Tumor/Lung	0.69 ± 0.15	1.07 ± 0.13	1.08 ± 0.25	1.45 ± 0.22
Tumor/Muscle	1.80 ± 0.48	2.29 ± 0.34	1.78 ± 0.69	2.79 ± 0.55

Table 2.

Selected biodistribution data and tumor-to-background ratios of ^99mTc-3P-RGD₂ in BALB/c nude mice (n = 5) bearing U87MG human glioma xenografts. The tumor uptake (%ID/g) is reported as an average plus/minus the standard deviation.

Organ	5 min	30 min	60 min	120 min
Blood	5.41 ± 0.53	1.54 ± 0.14	0.87 ± 0.10	0.32 ± 0.06
Brain	0.43 ± 0.08	0.41 ± 0.17	0.41 ± 0.05	0.23 ± 0.09
Eyes	2.74 ± 0.55	2.90 ± 22	2.57 ± 0.59	2.12 ± 0.86
Heart	6.39 ± 0.70	3.48 ± 0.45	3.13 ± 0.44	2.07 ± 0.75
Intestine	3.78 ± 0.52	4.24 ± 1.54	5.45 ± 1.47	3.37 ± 1.32
Kidneys	43.70 ± 3.27	32.89 ± 2.94	24.63 ± 3.59	19.05 ± 2.60
Liver	10.20 ± 1.12	5.82 ± 0.79	4.83 ± 0.60	4.52 ± 0.76
Lungs	8.64 ± 1.44	4.24 ± 0.87	3.50 ± 1.01	2.39 ± 0.71
Muscle	3.15 ± 0.64	2.25 ± 0.48	1.50 ± 0.49	1.23 ± 0.21
Spleen	5.84 ± 0.67	5.03 ± 0.45	4.02 ± 0.70	3.80 ± 0.50
Tumor	5.22 ± 0.72	6.77 ± 1.30	4.62 ± 0.57	3.93 ± 0.63
Tumor/Blood	0.96 ± 0.05	4.33 ± 0.12	5.86 ± 2.21	12.03 ± 3.62
Tumor/Liver	0.51 ± 0.03	1.16 ± 0.54	1.10 ± 0.54	0.84 ± 0.10
Tumor/Lung	0.61 ± 0.08	1.52 ± 0.74	1.33 ± 0.52	1.74 ± 0.63
Tumor/Muscle	1.68 ± 0.16	1.85 ± 1.38	2.25 ± 1.56	3.09 ± 0.40

Figure 3.

Direct comparison of the uptake values of ^99mTc-3P-RGD₂ and ^99mTc-4P-RGD₃ in U87MG glioma tumors (A), kidneys (C) and intestine (D), along with their tumor/blood ratios (B) over the 2 h study period. The most significant difference between two radiotracers was their uptake in the intestine and kidneys. E: Representative microscopic fluorescence images of U87MG glioma tissues obtained from the athymic (high blood vessel density) and BALB/c (low blood vessel density) nude mice. In the overlay images, the green color indicates the presence of blood vessels, red color indicates the presence of β₃, and yellow color indicates the β₃ expressed on the tumor vasculature.

Integrin α_vβ₃ Specificity

Figure 4A shows the 60-min planar images of U87MG-bearing athymic nude mice administered with ^99mTc-4P-RGD₃ in the absence/presence of RGD₂ (350 µg/mouse). Co-injection of excess RGD₂ resulted in a significant reduction in the uptake of ^99mTc-4P-RGD₃ in both U87MG glioma tumors and normal organs. Figure 4B displays the selected 60-min biodistribution data of ^99mTc-4P-RGD₃ in the absence/presence of RGD₂. Co-injection of excess RGD₂ (350 µg/mouse) blocked the tumor uptake of ^99mTc-4P-RGD₃ (0.59 ± 0.28 %ID/g with RGD₂ vs. 5.17 ± 1.01 %ID/g without RGD₂). The normal organ uptake was also blocked by co-injection of excess RGD₂. Similar results were reported for ^99mTc-3P-RGD₂ [14] and ¹¹¹In-labeled cyclic RGD dimers and tetramers [23].

Figure 4.

Top: Planar images of the athymic nude mice bearing U87MG glioma xenografts administered with ^99mTc-4P-RGD₃ with/without excess RGD₂ at 60 min post-injection. Arrows indicate the presence of tumors. Bottom: Direct comparison of the 60-min biodistribution data for ^99mTc-4P-RGD₃ in BALB/c nude mice (n = 4) bearing U87MG glioma xenografts with/without excess RGD₂. Blocking experiments were designed to demonstrate the specificity of ^99mTc-4P-RGD₃ for α_vβ₃.

SPECT/CT

SPECT/CT imaging studies were performed on ^99mTc-4P-RGD₃ or ^99mTc-3P-RGD₂ in athymic nude mice bearing U87MG glioma xenografts. ^99mTc-3P-RGD₂ was used purely for comparison purposes. Figure 5 displays the 3D and transverse views of SPECT/CT images of the U87MG-bearing nude mice administered with ~37 MBq of ^99mTc-4P-RGD₃ or ^99mTc-3P-RGD₂. The tumors were clearly visualized by SPECT/CT with excellent tumor-to-background contrast with both ^99mTc-4P-RGD₃ and ^99mTc-3P-RGD₂. These data clearly showed that ^99mTc-4P-RGD₃ is as good as ^99mTc-3P-RGD₂ for detection of the tumors. This conclusion is supported by the biodistribution data (Tables 1 and 2).

Figure 5.

The 3D and transverse views of SPECT/CT images of the athymic nude mice (B = bladder; I = intestine; K = kidney and T = tumor) bearing U87MG glioma xenografts. Each animal was administered with ~37 MBq of ^99mTc radiotracer (~1.0 µg HYNIC-3P-RGD₂ or HYNIC-4P-RGD₃). All tumors were clearly visualized with excellent contrast using both radiotracers.

Monitoring α_vβ₃ Expression

Three animal models (U87MG, MDA-MB-435 and PC-3) were used to illustrate the ability of ^99mTc-4P-RGD₃ to monitor α_vβ₃ expression. Figure 6A compares transverse views of SPECT/CT images of the tumor-bearing mice administered with ^99mTc-4P-RGD₃. The tumor uptake values follow the general ranking order of U87MG > MDA-MB-435 >> PC-3. Figure 6B shows the overlay images of xenografted tumor tissues (U87MG, MDA-MB-435 and PC-3). The β₃ expression level (fluorescence density) was defined by the percentage of red-colored area over the total area. We found that the α_vβ₃ expression level followed the general trend: U87MG > MDA-MB-435 >> PC-3. Figure 6C shows a plot of the %ID/g tumor uptake (radioactivity density) of ^99mTc-4P-RGD₃ obtained from SPECT quantification and the α_vβ₃ expression levels (fluorescence density) in U87MG, MDA-MB-435 and PC-3 tumors, as determined from the quantification of fluorescent intensity. Apparently, there was a linear relationship between the %ID/g tumor uptake of ^99mTc-4P-RGD₃ and the α_vβ₃ expression levels with R² being 0.98.

Figure 6.

A: Transverse views of SPECT/CT images from the tumor-bearing mice administered with ~50 MBq of ^99mTc-4P-RGD₃. B: The overlay images of the xenografted U87MG, MDA-MB-435 and PC-3 tumor tissues. For β₃ staining (red), frozen tissue slices were incubated with a β₃ primary antibody followed by a DyLight 594-conjugated secondary antibody. For CD31 staining (green), slices were incubated with a CD31 primary antibody followed by a FITC-conjugated secondary antibody. In the overlay images, the green color indicates the presence of blood vessels, red color indicates the presence of β₃, and yellow color indicates the β₃ expressed on tumor neovasculature. C: Linear relationship between the tumor uptake of ^99mTc-4P-RGD₃ from SPECT image semi-quantification and the relative β₃ expression levels (as represented by the % fluorescent intensity of red color over the total fluorescent intensity in the area) on three different tumor tissues to illustrate the capability of ^99mTc-4P-RGD₃ to monitor the α_vβ₃ expression levels in tumor of different origin.

Metabolic Stability

Figure 7 shows typical radio-HPLC chromatograms of ^99mTc-4P-RGD₃ in saline before being injected into animal (A), in the urine at 30 min p.i. (B) and 120 min p.i. (C), and in the feces at 120 min p.i. (D). There were no metabolites detected in the urine sample over the 2 h study period for ^99mTc-4P-RGD₃. More than 95% of ^99mTc-4P-RGD₃ remained intact in the feces sample at 2 h p.i. The in vivo metabolic stability of ^99mTc-4P-RGD₃ was better than that of ^99mTc-3P-RGD₂, only 70% of which remained intact in the feces samples at 2 h p.i. [14].

Figure 7.

Typical radio-HPLC chromatograms for ^99mTc-4P-RGD₃ in the saline before injection, in the urine at 30 min p.i. and 120 min p.i., and in the feces at 120 min p.i.

DISCUSSION

Previously, we found that cyclic RGD peptide dimers with the PEG₄ linkers (e.g. 3P-RGD₂) are bivalent in binding to the α_vβ₃ because of their better tumor-targeting capability than that of RGD₂, as indicated by their higher α_vβ₃-binding affinity and better tumor uptake of their radiotracers [13–29]. We also found that multimeric peptides are not necessarily multivalent in binding to the α_vβ₃, and multimerization of RGD peptides often leads to more radioactivity accumulation in the α_vβ₃-positive organs [20,23]. ^99mTc-4P-RGD₃ represents the “missing link” between radiolabeled cyclic RGD peptide dimers and tetramers. In this study, we found that ^99mTc-4P-RGD₃ and ^99mTc-3P-RGD₂ shared almost identical tumor uptake (Figure 3A) and similar biodistribution properties (Tables 1 and 2). Increasing peptide multiplicity from 3P-RGD₂ to 4P-RGD₃ offers no advantages in the tumor-targeting capability once bivalency is achieved. Similar conclusion was made with ¹¹¹In-labeled RGD tetramers [23]. The α_vβ₃ specificity of ^99mTc-4P-RGD₃ has been demonstrated by blocking experiments (Figure 4). ^99mTc-4P-RGD₃ had higher uptake than ^99mTc-3P-RGD₂ in the intestines (Figure 3C) and kidneys (Figure 3D). However, ^99mTc-4P-RGD₃ has better metabolic stability (Figure 7) than ^99mTc-3P-RGD₂. The glioma tumors were clearly visualized by SPECT/CT with excellent contrast with both ^99mTc-4P-RGD₃ and ^99mTc-3P-RGD₂ (Figure 6). Thus, ^99mTc-4P-RGD₃ is as good a SPECT radiotracer as ^99mTc-3P-RGD₂ for imaging α_vβ₃-positive tumors.

^99mTc-4P-RGD₃ exhibited higher uptake in the intestines, liver and kidneys than the tumor (Table 1). However, these organs are not visualized in SPECT/CT image (Figure 5). Similar results were reported for ^99mTc-3P-RGD₂ in various tumor-bearing models [37–40]. We also found that the radioactivity accumulation in these organs could be different even in the same animal over a 35-day period [37]. One possible explanation for this discrepancy is that ^99mTc-4P-RGD₃ has higher tumor uptake (8 – 12 %ID/g) in the U87MG glioma xenografted in the athymic nude mice used for SPECT imaging. Alternatively, this discrepancy might be caused by different amounts of HYNIC-4P-RGD₃ co-injected with ^99mTc-4P-RGD₃. For biodistribution, ~2 ng HYNIC-4P-RGD₃ was co-injected into each tumor-bearing mouse. For SPECT imaging, ~1 µg of HYNIC-4P-RGD₃ was co-administered with ^99mTc-4P-RGD₃, and could cause partial blocking of ^99mTc-4P-RGD₃ uptake in the organs (e.g. intestines, liver and kidneys) with limited total α_vβ₃ integrin population, as illustrated by β₃-staining of normal organ tissues [23]. The same explanation may also apply to ^99mTc-3P-RGD₂.

It is well-established that the α_vβ₃ expression on both tumor cells and neovasculature contributes to the total tumor uptake of an α_vβ₃-targeted radiotracer regardless of the peptide multiplicity [20,23]. In our previous studies, we found that the percentage of contribution from the tumor neovasculature depends on the tumor type (e.g. U87MG vs. PC-3) [21,26]. The contribution from tumor neovasculature to the total tumor uptake of ^99mTc-3P-RGD₂ is ~60% in the U87MG tumors xenografted in athymic nude mice [38]. In this study, we found that the tumors xenografted in the BALB/c nude mice had less uptake of ^99mTc-3P-RGD₂ than those in the athymic nude mice. Since they share the same U87MG glioma cells, this difference is caused by the lower α_vβ₃ expression levels (Figure 3) on the neovasculature of U87MG tumors xenografted in BALB/c nude mice, as determined by the IHC staining.

If the α_vβ₃–targeted radiotracer is used for accurate measurement of α_vβ₃ expression, there must be a linear relationship between the tumor uptake (radioactivity density) and α_vβ₃ levels (receptor density). The linear relationship between the %ID/g tumor uptake of ^99mTc-4P-RGD₃ and α_vβ₃ expression levels (Figure 6) clearly demonstrates its capability for noninvasive monitoring of the α_vβ₃ expression in tumor of different origin. In clinical settings, this capability is important for selection of the appropriate cancer patients who will benefit most from anti-α_vβ₃ and antiangiogenesis therapy. For example, if the cancer patient has high tumor uptake of ^99mTc-4P-RGD₃, he/she would be likely responsive to the anti-α_vβ₃ treatment. Conversely, if the cancer patient shows little tumor uptake of ^99mTc-4P-RGD₃, the therapy would not be effective regardless of the amount of anti-α_vβ₃ drug administered to the cancer patient.

CONCLUSION

In conclusion, ^99mTc-4P-RGD₃ could be prepared in high radiochemical purity with specific activity. ^99mTc-4P-RGD₃ and ^99mTc-3P-RGD₂ shared almost identical tumor uptake and similar biodistribution properties. Even though ^99mTc-4P-RGD₃ shows higher uptake in the intestines and kidneys, it has better metabolic stability than ^99mTc-3P-RGD₂. ^99mTc-4P-RGD₃ is as good a radiotracer as ^99mTc-3P-RGD₂ for tumor imaging and noninvasive monitoring of the α_vβ₃ expression.

ABBREVIATIONS

HYNIC-OSu

sodium succinimidyl 6-(2-(2-sulfonatobenzaldehyde)hydrazono)nicotinate)

ITLC

instant thin layer chromatography

MALDI

matrix-assisted laser desorption ionization

PET

positron emission tomography

RCP

radiochemical purity

SPECT

single photon emission computed tomography

RGD₂

E[c(RGDfK)]₂ = Glu[cyclo(Arg-Gly-Asp-D-Phe-Lys)]₂

RGD₄

E{E[c(RGDfK)]₂}₂ = Glu{E[cyclo(Arg-Gly-Asp-D-Phe-Lys)]₂}₂

2P-RGD₂

E[PEG₄-c(RGDfK)]₂ = Glu[cyclo[Arg-Gly-Asp-D-Phe-Lys(PEG₄)]]₂ (PEG₄ = 15-amino-4,7,10,13-tetraoxapentadecanoic acid)

3P-RGD₂

PEG₄-E[PEG₄-c(RGKfD)]₂ = PEG₄-Glu[cyclo[Arg-Gly-Asp-D-Phe-Lys(PEG₄)]]₂

4P-RGD₃

PEG₄-ACHDA[cyclo[Arg-Gly-Asp-D-Phe-Lys(PEG₄)]]₃ (ACHDA = 4-amino-4-(2-carboxyethyl)heptanedioic acid)

HYNIC-4P-RGD₃

HYNIC-PEG₄-ACHDA[cyclo[Arg-Gly-Asp-D-Phe-Lys(PEG₄)]]₃ (HYNIC = 6-(2-(2-sulfonatobenzaldehyde)hydrazono)nicotinyl)

¹⁸F-Alfatide-II

[¹⁸F]AlF(NOTA-2P-RGD₂) (NOTA = 1,4,7-tritazacyclononane-1,4,7-triacetic acid; and)

^99mTc-3P-RGD₂

[^99mTc(HYNIC-3P-RGD₂)(tricine)(TPPTS)] (TPPTS = trisodium triphenylphosphine-3,3',3"-trisulfonate)

^99mTc-4P-RGD₃

[^99mTc(HYNIC-4P-RGD₃)(tricine)(TPPTS)]