^99mTc-Galacto-RGD₂: A Novel ^99mTc-Labeled Cyclic RGD Peptide Dimer Useful for Tumor Imaging

Abstract

This study sought to evaluate [^99mTc(HYNIC-Galacto-RGD₂)(tricine)(TPPTS)] (^99mTc-Galacto-RGD₂: HYNIC = 6-hydrazinonicotinyl; Galacto-RGD₂ = Glu[cyclo[Arg-Gly-Asp-D-Phe-Lys(SAA-PEG₂-(1,2,3-triazole)-1-yl-4-methylamide)]]₂ (SAA = 7-amino-L-glycero-L-galacto-2,6-anhydro-7-deoxy-heptanamide, and PEG₂ = 3,6-dioxaoctanoic acid); and TPPTS = trisodium triphenylphosphine-3,3',3"-trisulfonate) as a new radiotracer for tumor imaging. Galacto-RGD₂ was prepared via the copper (I)-catalyzed 1,3-dipolar azide–alkyne Huisgen cycloaddition. HYNIC-Galacto-RGD₂ was prepared by reacting Galacto-RGD₂ with sodium succinimidyl 6-(2-(2-sulfonatobenzaldehyde)hydrazono)nicotinate (HYNIC-OSu) in the presence of diisopropylethylamine, and was evaluated for its integrin α_vβ₃ binding affinity against ¹²⁵I-echistatin bound to U87MG glioma cells. The IC₅₀ value for HYNIC-Galacto-RGD₂ was determined to be 20 ± 2 nM. ^99mTc-Galacto-RGD₂ was prepared in high specific activity (~185 GBq/µmol) and high radiochemical purity (>95%), and was evaluated in athymic nude mice bearing U87MG glioma xenografts for its tumor-targeting capability and biodistribution. The tumor uptake of ^99mTc-Galacto-RGD₂ was 10.30 ± 1.67, 8.37 ± 2.13, 6.86 ± 1.33 and 5.61 ± 1.52 %ID/g at 5, 30, 60 and 120 min p.i., respectively, which was in agreement with high integrin α_vβ₃ expression on glioma cells and neovasculature. Its lower uptake in intestines, lungs and spleen suggests that ^99mTc-Galacto-RGD₂ has advantages over ^99mTc-3P–RGD₂ ([^99mTc(HYNIC-3P–RGD₂)(tricine)(TPPTS)]: 3P–RGD₂ = PEG₄-E[PEG₄-c(RGDfK)]₂; PEG₄ = 15-amino-4,7,10,13-tetraoxapentadecanoic acid) for imaging tumors in the chest and abdominal regions. U87MG tumors were readily detected by SPECT and the tumor uptake of ^99mTc-Galacto-RGD₂ was integrin α_vβ₃-specific. ^99mTc-Galacto-RGD₂ also had very high metabolic stability. On the basis of results from this study, it was concluded that ^99mTc-Galacto-RGD₂ is an excellent radiotracer for imaging integrin α_vβ₃-positive tumors and related metastases.

INTRODUCTION

Integrin α_vβ₃ plays a significant role in tumor angiogenesis and metastasis.^1–8 It is a receptor for the extracellular matrix proteins (e.g.vitronectin, fibronectin, fibrinogen, lamin, collagen, Von Willibrand’s factor and osteoponin) with the exposed arginine-glycine-aspartic (RGD) peptide sequence.^9–11 Integrin α_vβ₃ is expressed at low levels on epithelial cells and mature endothelial cells, but it is overexpressed on activated endothelial cells of neovasculature and some tumor cells. Thus, integrin α_vβ₃ is considered an interesting molecular target for early cancer detection.^12–18

Cyclic RGD peptides, such as E[c(RGDfK)]₂ (Figure 1: RGD₂) and E{E[c(RGDfK)]₂}₂ (RGD₄), are potent integrin α_vβ₃ receptor antagonists.^10,11,16,18 Over the last several years, many radiolabeled (^99mTc, ¹⁸F, ⁶⁴Cu, ⁶⁸Ga and ¹¹¹In) multimeric cyclic RGD peptides have been evaluated as integrin α_vβ₃-targeted radiotracers for tumor imaging by single photon emission computed tomography (SPECT) or positron emission tomography (PET).^19–39 It was shown that these cyclic RGD peptides were able to bind integrin α_vβ₃ with high specificity.^16,18 Multiple RGD moieties were utilized to maximize their integrin α_vβ₃ binding affinity and the radiotracer tumor uptake.^15,16,18 It was found that radiolabeled multimeric RGD peptides had significantly higher tumor uptake with much longer tumor retention time than their monomeric counterparts.^{16,18,24–39} It was also found that cyclic RGD peptide tetramers, such as RGD₄, are bivalent in binding to integrin α_vβ₃ even though they have four identical RGD motifs.^36,38

Figure 1.

The structure of cyclic RGD peptides (c(RGDfK), RGD₂, 3P–RGD₂, and Galacto-RGD₂) and their corresponding ternary ligand complexes [^99mTc(HYNIC-BM)(tricine)(TPPTS)] (BM = biomolecule; ^99mTc-RGD₂: BM = RGD₂; ^99mTc-3P–RGD₂: BM = 3P–RGD₂^99mTc-Galacto-RGD₂: BM = Galacto-RGD₂. The 7-amino-L-glycero-L-galacto-2,6-anhydro-7-deoxyheptanamide (SAA) and 1,2,3-triazole moieties are used to increase the hydrophilicity of ^99mTc-Galacto-RGD₂, and minimize its accumulation in normal organs, such as intestines, lungs and spleen.

We have been interested in radiolabeled cyclic RGD peptides as PET or SPECT radiotracers for molecular imaging of solid tumors.^30–44 [^99mTc(HYNIC-3P–RGD₂)(tricine)(TPPTS)] (Figure 1: ^99mTc-3P–RGD₂: HYNIC = 6-hydrazinonicotinyl; 3P–RGD₂ = PEG₄-E[PEG₄-c(RGDfK)]₂; PEG₄ = 15-amino-4,7,10,13-tetraoxapentadecanoic acid; and TPPTS = trisodium triphenylphosphine-3,3',3"-trisulfonate) is a ^99mTc-labeled cyclic RGD peptide dimer.³¹ It could be prepared from ^99mTcO₄^‒ with high specific activity using a kit formulation.^31,40.^99mTc-3P–RGD₂ showed very high tumor uptake and rapid renal clearance in glioma-bearing nude mice,³¹ and its tumor uptake correlated well linearly with the integrin α_vβ₃ expression levels in five different tumor-bearing small animal models.⁴². ^99mTc-3P–RGD₂ SPECT/CT has been successfully used to quantify the tumor uptake and for noninvasive monitoring of progression of breast cancer lung metastases.^43,44 It is currently under clinical investigation as a new radiotracer for tumor imaging in cancer patients.^45,46

However, ^99mTc-3P–RGD₂ has high uptake in intestines and spleen,^31,45,46 which makes it difficult to image tumors in the abdominal region. To minimize excess radioactivity accumulation in normal organs, we designed and prepared a new cyclic RGD peptide dimer, Galacto-RGD₂ (Figure 1), in which the 7-amino-L-glycero-L-galacto-2,6-anhydro-7-deoxyheptanamide (SAA) groups were used to bridge the two cyclic RGD moieties. SAA has been successfully used to improve pharmacokinetics of ¹⁸F-labeled cyclic RGD peptides.^19–21,47 The 1,2,3-triazole moiety was reported to have beneficial effect on pharmacokinetics of radiotracers by increasing their washout efficiency from liver and GI tract.⁴⁸ With this in mind, we decided to combine the SAA and 1,2,3-triazole moieties in Galacto-RGD₂. Our hypothesis is that the SAA and 1,2,3-triazole moieties will help to reduce its accumulation in normal organs, such as the intestines, liver, lungs and spleen.

In this report, we present the synthesis of Galacto-RGD_2, and evaluations of its ^99mTc complex [^99mTc(HYNIC-Galacto-RGD₂)(tricine)(TPPTS)] (Figure 1: ^99mTc-Galacto-RGD₂) as a new radiotracer for tumor imaging. Integrin α_vβ₃ binding affinity of HYNIC-Galacto-RGD₂ was determined in an in vitro displacement assay. Biodistribution properties of ^99mTc-Galacto-RGD₂ were evaluated in the athymic nude mice bearing U87MG glioma xenografts, and compared to those of ^99mTc-3P–RGD₂. The main objective of this study was to demonstrate that SAA and 1,2,3-triazole moieties are indeed able to improve excretion kinetics of ^99mTc radiotracer from non-cancerous organs.

EXPERIMENTAL SECTION

Materials and Instruments

Chemicals were purchased from Sigma/Aldrich (St. Louis, MO), and were used without purification unless specified. The Boc-protected glutamic acid was purchased from Senn Chemicals (San Diego, CA), and 8-azido-3,6-dioxaoctanoic acid (N₃-PEG₂-OH) was from Peptides International Inc. (Louisville, KY). The protected peptide [cyclo[Arg(Pbf)-Gly-Asp(OtBu)-D-Phe-Lys(SAA)]] (Galacto-RGD) was prepared using the procedure reported in the literature.⁴⁷ Sodium succinimidyl 6-(2-(2-sulfonatobenzaldehyde)hydrazono)nicotinate (HYNIC-OSu) was prepared according to the literature method.⁴⁹ [^99mTc(HYNIC-3P–RGD₂)(tricine)(TPPTS)] (^99mTc-3P–RGD₂) was prepared using the procedure described in our previous reports.^31,40 Na^99mTcO₄ was obtained from Cardinal HealthCare® (Chicago, IL). The MALDI (matrix-assisted laser desorption ionization) mass spectral data were collected on an Applied Biosystems Voyager DE PRO mass spectrometer (Framingham, MA), the Department of Chemistry, Purdue University. Melting point was determined on a Buchi B-540 melting point apparatus and was uncorrected. Optical rotation was determined on a DIP-370 polarimeter (Jasco, Easton, MD). Thin layer chromatography was performed using Merck F₂₅₄ silica gel plates (EMD, Darmstadt, Germany). NMR spectra were recorded with a Varian Unity 500 Plus spectrometer (Palo Alto, CA) in DMSO-d₆.

HPLC Methods

The RP-HPLC (Method 1) used a Shimadzu preparative system consisting of two LC-8A pumps controlled by a Shimadzu SCL-10A system, and a Shimadzu UV/VIS detector SPD-6A (λ=220 nm) equipped with a preparative flow cell, a Prochrom preparative HPLC column (50 mm × 35 cm,100 Å pore size, Phenomenex, Torrance, CA). The flow rate was 100 mL/min with a gradient phase starting from 90% A (0.05 % TFA in H₂O) and 10 % B (0.05 % TFA in CH₃CN) at 0 min to 60%A and 40 % B at 60 min. HPLC Method 2 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 70%A and 30% B at 20 min. The radio-HPLC (Method 3) used the LabAlliance HPLC system equipped with a β-ram IN/US detector (Tampa, FL) and Zorbax C₁₈ 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 60% A and 40% B at 20 min.

Boc-L-Glutamic Acid Bis-Propargyl Amide (1)

To a solution of Boc-glutamic acid (2.47 g, 10 mmol) and propargylamine hydrochloride (2.11 g, 23 mmol) in DMF (30 mL) were added diphenylphosphoryl azide (DPPA: 5.2 mL, 24 mmol) and triethylamine (Et₃N, 3.2 mL, 23 mmol) at 0 °C under vigorous stirring. The reaction was allowed to proceed overnight at 0 °C. The pH value of the reaction mixture was kept at 7 ~ 8 with Et₃N. The reaction mixture was taken into EtOAc (500 mL) and washed with 0.2 N HCl (2 × 200 mL), brine (100 mL), saturated NaHCO₃ solution (3 × 200 mL), and brine (2 × 100 mL). After drying over Na₂SO₄, EtOAc was removed to yield 4.16 g of crude product, which upon trituration with hot isopropyl ether afforded a white solid (2.66 g, 82.9 %). Rf (EtOAc/Hexane/AcOH 2:1:0.01) = 0.20, melting point 146.2–146.5°C, [α] _D²⁵ 10.4° (c = 1, CHCl₃). ESI-MS: m/z = 344.2 [M + Na]⁺; 322.2 [M + H]⁺. ¹H NMR (DMSO-d6, chemical shift δ in ppm, and atom labels according to the structure below): δ 8.25 [1H, bt, J=5.6 Hz, N(1a)-H], 8.24 [1H, bt, J=5.6 Hz, N(5a)-H], 6.88 [1H, d, J= 8.0 Hz, N(2a)-H], 3.87 [1H, m, C(2)-H], 3.85 [2H, dd, J=5.6 Hz, J=2.3 Hz, C(1b)-H₂], 3.82 [2H, dd, J=5.6 Hz, J=2.6 Hz, C(5b)-H₂], 3.10 [1H, t, J=2.3 Hz, C(1d)-H], 3.08 [1H, t, J=2.6 Hz, C(5d)-H], 2.10 [2H, m, C(4)-H₂], 1.82 [1H, m, C(3)- H₂], 1.67 [1H, m, C(3)- H₂], and 1.37 [9H, s, C(2e)-H3]. ¹³C NMR (DMSO-d6, chemical shift δ in ppm): 171.5 (C-1), 171.2 (C-5), 155.2 (C-2b), 81.2 (C-1c), 81.1 (C-5c), 78.1 (C-2d), 73.0 (C-5d), 72.9 (C-1d), 53.8 (C-2), 31.6 (C-4), 28.2 (C-2e), 27.9 (C-1b), 27.8 (C3 and C-5b). Anal. Calcd for C₁₆H₂₃N₃O₄: C, 59.80; H, 7.21; N, 13.08. Found: C, 59.78; H, 7.19; N, 13.00.

Cyclo[Arg(Pbf)-Gly-Asp(OtBu)-D-Phe-Lys(SAA-PEG2-N3)] (2)

To a solution of cyclo[Arg(Pbf)-Gly-Asp(OtBu)-D-Phe-Lys(SAA)] (1.10 g, 1.0 mmol) and N₃-PEG₂-OH (0.28 g, 1.48 mmol) in DMF (15 mL) was added DPPA (0.43 mL, 2.0 mmol) at 0 °C with stirring, followed by the addition of Et₃N (0.21 mL, 1.51 mmol). The reaction mixture was kept at 0 °C for 25 h, with occasional adjustments of the pH (7 – 8), with Et3N. After the reaction, the mixture was diluted with 0.4 N HCl (100 mL) and extracted with EtOAc (3 × 300 mL). The combined organic extracts were concentrated. The precipitated product was collected by filtration, washed with cold Et₂O, and dried in vacuo to afford 0.81 g of 2 in 63.7 % yield as a colorless solid. ESI-MS: m/z = 945.55 [M + H]⁺ (M = 945.43 calcd. for [C₄₀H₅₉N₁₃O₁₄]).

Boc-Glu[cyclo[Arg(Pbf)-Gly-Asp(OtBu)-D-Phe-Lys(SAA-PEG2-(1,2,3-triazole)-1-yl-4-methylamide)]]2 (3)

To a stirred solution of 1 (94 mg, 0.29 mmol) and 2 (750 mg, 0.59 mmol) in a mixture of H₂O (2.9 mL) and t-BuOH (5.8 mL) were added 1.73 mL of 0.2 M CuSO₄ 5H₂O and 1.73 mL of 0.5 M sodium ascorbate solution at room temperature. The mixture was stirred for 70 min, then diluted with 60 mL 1-BuOH and 40 mL H₂O. The organic layer was washed with H₂O (2 × 30 mL) and concentrated under reduced pressure. Addition of Et₂O resulted in a precipitate, which was collected by filtration, washed with Et₂O, and dried to give 760 mg of 3. The yield was 91.5 %. ESI-MS: m/z = 2249.05 [M+H]⁺ (M = 2249.05 calcd. for [C₉₆H₁₄₅N₂₉O₃₄]).

H-Glu[cyclo[Arg-Gly-Asp-D-Phe-Lys(SAA-PEG₂-(1,2,3-triazole)-1-yl-4-methylamide)]]₂ (Galacto-RGD₂)

The protected Galacto-RGD₂ (750 mg, 0.26 mmol) was dissolved in 20 mL of a solution TFA/TIPS/H₂O (95/2.5/2.5, v/v/v) at room temperature. After 2 h, the reaction mixture was concentrated under reduced pressure. Crude Galacto-RGD₂ was precipitated with cold Et₂O, collected by centrifugation, washed with Et₂O (3 × 30 mL), and dried in vacuo to give 690 mg of white solid, which was purified by RP-HPLC (Method 1) to yield Galacto-RGD₂ (105 mg, 16.2 %) as a TFA salt. The HPLC purity was 99.3 %. ESI-MS: m/z = 2149.12 [M+H]⁺ (M = 2149.05 calcd. for [C₉₁H₁₃₇N₂₉O₃₄]).

HYNIC-Glu[cyclo[Arg-Gly-Asp-D-Phe-Lys(SAA-PEG₂-(1,2,3-triazole)-1-yl-4-methylamide)]]₂ (HYNIC-Galacto-RGD₂)

HYNIC-OSu (5.4 mg, 12 µmol) and Galacto-RGD₂ (6.5 mg, 3 µmol) were dissolved in DMF (2 mL). After addition of excess diisopropylethylamine (DIEA, 50 µmol), the reaction mixture was stirred for ~10 days at room temperature. To the reaction mixture was added 2 mL of water after completion of conjugation. The pH value was then adjusted to 3 – 4 using TFA. The product was separated from the reaction mixture by HPLC (Method 2). Fractions at 14.5 min were collected. Lyophilization of collected fractions afforded HYNIC-Galacto-RGD_2. The yield was 3.5 mg (~48%). MALDI-MS: m/z = 2452.3 for [M + H]⁺ (M = 2451.03 calcd. for [C₁₀₄H₁₄₆N₃₂O₃₆S]).

^99mTc-Labeling

To a lyophilized vial containing 20 µg HYNIC-Galacto-RGD₂, 7 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 MBq in saline). The reconstituted vial was then heated in a boiling water bath for ~30 min. After radiolabeling, a sample of the resulting solution was analyzed by radio-HPLC (Method 3). The radiochemical purity was >90% for imaging and biodistribution studies. The solution stability was monitored by radio-HPLC for 24 h.

Dose Preparation

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

Tumor Cell Culture

The U87MG cell line was obtained from ATCC (American Type Culture Collection, Manassas, VA). U87MG cells were cultured in the Minimum Essential Medium, Eagle with Earle’s Balanced Salt Solution (non-essential amino acids sodium pyruvate), and were supplemented with 10% fetal bovine serum (FBS, ATCC) and 1% penicillin and streptomycin solution 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 to maintain exponential growth.

In Vitro Whole-Cell Integrin α_vβ₃ Binding Assay

The integrin binding affinity of cyclic RGD peptides were assessed via a cellular competitive displacement assay using¹²⁵ I-echistatin (Perkin Elmer, Branford, CT) as the integrin-specific radioligand.^31,32 Briefly, the filter multiscreen DV plates (Millipore, Billerica, MA) were seeded with 1×10⁵ U87MG cells in binding buffer (20 mM Tris, 150 mM NaCl, 2 mM CaCl₂, 1 mM MnCl₂, 1 mM MgCl₂, 0.1% (wt/vol) bovine serum albumin; and pH 7.4) and ¹²⁵I-echistatin (0.75 – 1.0 kBq) in the presence of increasing concentrations of the cyclic RGD peptide, incubated for 2 h at room temperature. After removing unbound ¹²⁵I-echistatin, the hydrophilic PVDF filters were washed 3× with the binding buffer, and then collected. Radioactivity was determined using a Perkin Elmer Wizard – 1480 γ-counter (Shelton, CT). Experiments were carried out twice with triplicates. IC₅₀ values were calculated by fitting experimental data with nonlinear regression using GraphPad Prism™ (GraphPad Software, Inc., San Diego, CA), and were reported as an average plus/minus standard deviation.

Animal Models

Biodistribution and 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). Female athymic nu/nu mice (4 – 5 weeks) were purchased from Harlan (Indianapolis, IN), and were inoculated subcutaneously with 5 × 10⁶ of U87MG cells into the shoulder flank of each animal. Four weeks after inoculation, the tumor size was 0.1 – 0.5 g, and animals were used for biodistribution and imaging studies.

Biodistribution Protocol

The tumor-bearing mice (20 – 25 g) were randomly selected, and each animal was administered with ~0.1 MBq of ^99mTc-Galacto-RGD₂ (~2 ng HYNIC-Galacto-RGD₂) by tail vein injection. Animals (5 – 8) were sacrificed by sodium pentobarbital overdose (~200 mg/kg) at 5, 30, 60 and 120 min post-injection (p.i.). Blood, tumors and normal organs (brain, eyes, heart, spleen, lungs, liver, kidneys, muscle and intestine) were harvested, washed with saline, dried with absorbent tissue, weighed, and counted on a Perkin Elmer Wizard – 1480 γ-counter. The organ uptake was calculated as the percentage of injected dose per gram of wet tissue (%ID/g). The blocking experiment was performed using RGD₂ as the blocking agent in six animals. Each animal was administered with ~0.1 MBq of ^99mTc-Galacto-RGD₂ along with ~350 µg (~14 mg/kg) of RGD₂. Biodistribution data (%ID/g) and tumor-to-background (T/B) ratios were expressed as the average plus standard deviation.

SPECT/CT Imaging

SPECT/CT images were obtained using a u-SPECT-II/CT scanner (Milabs, Utrecht, The Netherlands) equipped with a 0.6 mm multi-pinhole collimator. The glioma-bearing mouse was injected with ~37 MBq of ^99mTc-Galacto-RGD₂ (~0.8 µg HYNIC-Galacto-RGD₂) 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 Reconstruction and 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. The tumor delineation was performed on CT and SPECT images according to the literature method.^43,44 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-Galacto-RGD₂ was expressed the percentage of injected dose per unit volume (%ID/cm³), and was compared with the data from biodistribution in the same tumor-bearing animal model.

Metabolism

Athymic nude mice (n = 3) without tumors were used for metabolism study. Each animal was administered with ~10 MBq of ^99mTc-Galacto-RGD₂ via the tail vein. The urine samples were collected at 30 min and 120 min p.i. by manual void, and were mixed with equal volume of 50% acetonitrile aqueous solution. The mixture was centrifuged at 8,000 rpm. The supernatant was collected and filtered with a 0.20 µm Millex-LG filter to remove foreign particles. The filtrate was analyzed by HPLC (Method 2). Feces samples were collected at 120 min p.i. and suspended in 20% acetonitrile aqueous solution after homogenization. The resulting mixture was vortexed for ~5 min. After centrifuging at 8,000 rpm, the supernatant was collected and filtered with a 0.20 µm Millex-LG filter to remove foreign particles. The filtrate was then analyzed by HPLC (Method 2). The percentage of radioactivity recovery was >95% (by γ-counting) for both urine and feces samples.

Cultured Primary Tumor Cells

The xenografted U87MG tumors were harvested, and were immersed in a growth medium immediately after gamma counting. Tumor tissues were rinsed twice with the Hanks’ solution and dissected into small pieces with razor blades. Small tumor pieces were digested with 0.25% Trypsin (1 mM EDTA, without Ca²⁺ and Mg²⁺) solution at 37 °C for 30 min, followed by vigorous pipetting and filtration through a 40 µm mesh nylon screen. After centrifugation at 1000 rpm for 5 min, the pellet was re-suspended in the culture media and incubated at 37 °C in a humidified atmosphere containing 5% CO₂. The purity of tumor cells could reach ~90% after two passages, and the third passage was used for cellular immunostaining studies.

Cellular Immunostaining

The primary U87MG glioma cells were seeded into 8-well chamber slides, and allowed to spread for >24 h. Cells were fixed in −20 °C methanol for 5 min and rinsed with PBS. Cells were incubated in 5% BSA for 30 min to block nonspecific binding, and then added Alexa Fluor 488 conjugated mouse anti-human integrin α_vβ₃ monoclonal antibody LM609 (1:100, Millipore, Billerica, MA) for 1 h at room temperature, or incubated with rabbit anti-human and murine β₃ antibody followed by incubation with Alexa Fluor 594 conjugated goat anti-rabbit IgG (Santa Cruz, CA). After washing with PBS buffer, the slides were mounted with Dapi-Fluoromount-G (SouthernBiotech, Birmingham, AL). The fluorescence was visualized with an Olympic BX51 microscope (Olympus America Inc., Center Valley, PA).

Tumor Tissue Immunohistochemistry

The U87MG tumors harvested from tumor-bearing mice were immediately snap-frozen in the OCT (optical cutting temperature) solution, and were then cut into slices (5 µm). After thorough drying at room temperature, slices were fixed with ice-cold acetone for 10 min, and dried in the air for 20 min. Sections were blocked with 10% goat serum for 30 min at room temperature, and then were incubated with the hamster anti-integrin β₃ antibody (1:100, BD Biosciences, San Jose, CA) and rat anti-CD31 antibody (1:100, BD Biosciences) for 1 h at room temperature. After incubating with the Cy3-conjugated goat anti-hamster and fluorescein isothiocyanate (FITC)-conjugated goat anti-rat secondary antibodies (1:100, Jackson ImmunoResearch Inc., West Grove, PA) and washing with PBS, the fluorescence was visualized with an Olympus fluorescence microscope (Olympus America Inc., Center Valley, PA). All the pictures were taken under 200x magnification with the same exposure time. Brightness and contrast adjustments were made equally to all images.

Statistical Analysis

All experimental data were expressed as the average plus/minus 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.

RESULTS

Synthesis of Galacto-RGD₂

Compound 1, a key intermediate for preparation of Galacto-RGD₂, was prepared (Chart I: ~83% yield) from the reaction of Boc-glutamic acid with two equivalents of propargylamine in the presence of DPPA, which is a coupling agent with low risk of racemization. ^50,51 DPPA was also employed in the preparation of 2. Synthesis of 3 was accomplished in >90% yield by the Cu(I)-catalyzed 1,3-dipolar azide–alkyne Huisgen cycloaddition. It was not necessary to protect the hydroxyl groups in the SAA moieties during this reaction. Deprotection of the Pbf and t-Butyl protecting groups in 3 under acidic conditions resulted in Galacto-RGD₂, which was characterized by HPLC (Figure SI1), ESI-MS (Figure SI2) and NMR (Figures SI3 – SI8: DQF-COSY, NOESY, ROESY, TOCSY, HSQC, and HMBC). The ESI-MS and NMR data (Tables SI1 and SI2) were completely consistent with the structure and composition proposed for Galacto-RGD₂.

Chart I.

Syntheses of Galacto-RGD₂

Synthesis of HYNIC-Galacto-RGD₂

HYNIC-Galacto-RGD₂ was prepared from the reaction of Galacto-RGD₂ with HYNIC-OSu in the presence of excess DIEA. The conjugation reaction was slow, and took about 10 days to complete at room temperature. This was likely due to the fact that the primary amine in Galacto-RGD₂ is stereochemically “hindered” by two bulky Galacto-RGD monomers. HYNIC-Galacto-RGD₂ was purified by semi-prep HPLC and characterized by ESI-MS. Its HPLC purity was > 95% before being used for ^99mTc-labeling. ESI-MS data was consistent with the proposed composition for HYNIC-Galacto-RGD₂.

Integrin α_vβ₃ Binding Affinity

Figure 2 shows the displacement curves of ¹²⁵I-echistatin bound to U87MG human glioma cells in the presence of HYNIC-Galacto-RGD₂. For comparison purpose, we also evaluated c(RGDyK), HYNIC-RGD₂ and HYNIC-3P–RGD₂ in the same assay. IC₅₀ values were calculated to be 20±2, 32±1, 85±2 and 422±15 nM for HYNIC-Galacto-RGD_2, HYNIC-3P–RGD₂, HYNIC-RGD₂ and c(RGDyK), respectively. The IC₅₀ values of c(RGDyK) and HYNIC-RGD₂ were close to those from our previous report.^31,32 The integrin α_vβ₃ binding affinity follows the rank order of HYNIC-Galacto-RGD₂ ≥ HYNIC-3P–RGD₂ > HYNIC-RGD₂ >> c(RGDyK) using the same competitive displacement assay.

Figure 2.

The competitive displacement curves of ¹²⁵I-echistatin bound to U87MG human glioma cells in the presence of cyclic RGD peptides. HYNIC-3P–RGD_2, HYNIC-RGD₂ and c(RGDyK) were used for comparison purposes. IC₅₀ values were calculated to be 20±2, 32±1, 85±2 and 422±15 nM for HYNIC-Galacto-RGD_2, HYNIC-3P–RGD_2, HYNIC-RGD₂ and c(RGDyK), respectively.

Radiochemistry

^99mTc-Galacto-RGD₂ was prepared from the reaction of HYNIC-Galacto-RGD₂ with ^99mTcO₄^‒ in the presence of excess tricine and TPPTS. ^99mTc-labeling was accomplished by heating the reaction mixture at 100 °C for 30 min. Figure 2 shows representative radio-HPLC chromatograms of ^99mTc-Galacto-RGD₂ in the kit matrix at 1 and 24 h post-labeling. Its radiochemical purity for was >95% without further chromatographic purification. The specific activity was ~185 GBq/µmol. It remained stable in the kit matrix for more than 24 h post-labeling.

Biodistribution

Table 1 lists the biodistribution data of ^99mTc-Galacto-RGD₂ in athymic nude mice bearing U87MG glioma xenografts. Its tumor uptake was 10.30 ± 1.67, 8.37 ± 2.13, 6.86 ± 1.33 and 5.61 ± 1.52 %ID/g at 5, 30, 60 and 120 min p.i., respectively. For comparison purposes, we also obtained the 60-min biodistribution date of ^99mTc-3P–RGD₂ in the same tumor-bearing animal model. As illustrate in Figure 4A, the tumor uptake of ^99mTc-Galacto-RGD₂ was close to that of ^99mTc-3P–RGD₂ at <60 min p.i. However, its 120-min tumor uptake (5.61 ± 1.52 %ID/g) that was significantly lower than that of ^99mTc-3P–RGD₂ (9.74 ± 3.22 %ID/g).³¹ Replacing PEG₄ linkers in ^99mTc-3P–RGD₂ with a pair of SAA, 1,2,3-triazole and PEG₂ moieties resulted in faster tumor washout of ^99mTc-Galacto-RGD₂. The liver uptake of ^99mTc-Galacto-RGD₂ was 4.00 ±1.10, 2.82 ± 1.25, 3.07 ± 2.14, and 1.54 ±0.43%ID/g with the tumor/liver ratios being 2.66 ± 0.51, 3.25 ± 0.85, 3.05 ± 1.69 and 4.03 ± 2.04 at 5, 30, 60, and 120 min p.i., respectively. Its liver uptake was comparable to that of ^99mTc-3P–RGD₂ over 2 h period.³¹ The main difference between ^99mTc-Galacto-RGD₂ and ^99mTc-3P–RGD₂ was their uptake in intestines, lungs and spleen (Figure 4A). For example, the intestine uptake of ^99mTc-Galacto-RGD₂ (5.45 ± 1.47 %ID/g) was much lower (p<0.01) than that of ^99mTc-3P–RGD₂ (9.45 ± 1.31 %ID/g) at 60 min p.i.

Table 1.

Selected biodistribution data and tumor-to-background ratios of ^99mTc-Galacto-RGD₂ in athymic nude mice bearing U87MG human glioma xenografts. The tumor uptake was expressed as an average plus/minus the standard deviation.

Organ	5 min (n = 5)	30 min (n = 5)	60 min (n = 8)	120 min (n = 8)
Blood	1.10 ± 0.16	0.92 ± 0.33	0.56 ± 0.25	0.22 ± 0.03
Brain	0.41 ± 0.10	0.22 ± 0.06	0.35 ± 0.28	0.14 ± 0.12
Eyes	1.54 ± 0.49	1.20 ± 0.50	0.81 ± 0.39	0.52 ± 0.15
Heart	2.36 ± 0.29	1.65 ± 1.10	1.27 ± 0.61	0.56 ± 0.08
Intestine	3.78 ± 0.52	4.24 ± 1.54	5.45 ± 1.47	3.37 ± 1.32
Kidney	18.75 ± 2.79	13.13 ± 4.75	11.70 ± 4.06	6.04 ± 1.92
Liver	4.00 ± 0.10	2.82 ± 1.25	3.07 ± 2.14	1.54 ±0.43
Lungs	5.41 ± 0.58	4.24 ± 1.71	2.93 ± 0.94	1.31 ± 0.23
Muscle	2.73 ± 0.89	1.10 ± 0.25	0.88 ± 0.34	0.47 ± 0.12
Spleen	2.97 ± 0.43	2.53 ± 0.85	1.99 ± 0.71	1.46 ± 0.26
Tumor	10.30 ± 1.67	8.37 ± 2.13	6.86 ± 1.33	5.61 ± 1.52
Tumor/Blood	9.43 ± 1.19	9.65 ± 2.03	15.94 ± 8.85	26.48 ± 8.71
Tumor/Kidney	0.52 ± 0.14	0.61 ±0.12	0.79 ±0.40	0.89 ±0.48
Tumor/Liver	2.66 ± 0.51	3.25 ± 0.85	3.05 ± 1.69	4.03 ± 2.04
Tumor/Lung	1.90 ± 0.21	2.19 ± 0.75	2.73 ± 1.33	4.38 ± 1.34
Tumor/Muscle	3.98 ± 0.75	7.97 ± 2.53	9.40 ± 5.11	12.91 ± 4.95

Figure 4.

A: Comparison of the 60-min biodistribution data between ^99mTc-Galacto-RGD₂ (n = 8) and ^99mTc-3P–RGD₂ (n = 6) in the athymic nude mice bearing U87MG glioma xenografts to demonstrate the advantage of ^99mTc-Galacto-RGD2 over ^99mTc-3P–RGD₂B: The selected 60-min biodistribution data of ^99mTc-Galacto-RGD₂ in the athymic nude mice (n = 8) bearing U87MG glioma xenografts with/without co-injection of RGD₂ (350 µg/mouse or 14 mg/kg) to demonstrate its integrin α_vβ₃ specificity.

Integrin α_vβ₃ Specificity

Figure 4B shows the 60-min biodistribution data of ^99mTc-Galacto-RGD₂ in athymic nude mice (n = 6) bearing U87MG glioma xenografts in the absence/presence of excess RGD2 (350 µg/mouse or 14 mg/kg). Co-injection of excess RGD₂ significantly blocked its tumor uptake (1.48 ± 0.42 %ID/g with RGD₂ vs. 6.86 ± 1.33 %ID/g without RGD₂). The normal organ uptake was also blocked by co-injection of excess RGD₂. For example, the uptake of ^99mTc-Galacto-RGD₂ in the intestine, lungs and spleen was 5.56 ± 1.73, 2.19 ± 0.94, and 1.09 ± 0.44 %ID/g, respectively, without RGD₂, while its uptake in the same organs was 1.82 ± 0.86, 1.52 ± 0.27, and 0.65 ± 0.07 %ID/g, respectively, in the presence of RGD₂. This experiment clearly demonstrated that the tumor uptake of ^99mTc-Galacto-RGD2 is integrin α_vβ₃-specific.

SPECT/CT Imaging

Figure 5A shows 3D (left) and transverse (right) views of SPECT/CT images of glioma-bearing mice administered with ~37 MBq of ^99mTc-Galacto-RGD₂ and ^99mTc-3P–RGD₂. ^99mTc-3P–RGD₂ was used for comparison purpose. Their tumor uptake was ~8.5 %ID/cm³ and ~7.0 %ID/cm³ , respectively, on the basis of SPECT quantification. The tumor was clearly visualized by SPECT/CT with excellent contrast with both radiotracers. As expected from biodistribution (Table 1),^99mTc-Galacto-RGD₂ and ^99mTc-3P–RGD₂ displayed substantial GI uptake (Figure 5A). The SPECT/CT data clearly showed that both are excellent radiotracers for imaging U87MG gliomas. Figure 5B shows cellular immunostaining data. Figure 5C displays microscopic images of selected tumor slice stained with anti-integrin β₃ (red color) or anti-CD31 (green color) antibody. CD31 was used as a biomarker for tumor blood vessels (mature and neovasculature), and was visualized with FITC (green). Integrin β₃ was visualized with Cy3 (red). Yellow color (overlay between red β₃ staining and green CD31 staining) indicates the presence of integrin α_vβ₃ on tumor neovasculature. It was clear that integrin α_vβ₃ was highly expressed on both U87MG glioma cells and neovasculature, which was in complete agreement with the high uptake of ^99mTc-Galacto-RGD₂ in glioma tumors (Table 1).

Figure 5.

A: The 3D and transverse views of SPECT/CT images of the athymic nude mice bearing U87MG human glioma xenografts. The animal was administered with ~37 MBq of ^99mTc-3P–RGD₂ or ^99mTc-Galacto-RGD₂. SPECT/CT study was designed to illustrate their utility for tumor imaging. B: Representative fluorescence microscopic images of the U87MG primary tumor cells, which were isolated from U87MG glioma tissue and stained with Alexa Fluor 488 conjugated mouse anti-human integrin α_vβ₃ monoclonal antibody LM609 (green) or with rabbit anti-human and murine integrin β₃ antibody (red). Blue color indicates the presence of nuclei stained with DAPI (4’,6-diamidino-2-phenylindole). C: Selected microscopic images (Magnification: 200x) of the tumor slice stained with hamster anti-integrin β₃ (red) and rat anti-CD31 (green) antibodies. Yellow color (red integrin β₃ staining merged with green CD31 staining) indicates the presence of α_vβ₃ on tumor neovasculature.

Metabolism

Metabolism study was performed to show the in vivo stability of ^99mTc-Galacto-RGD₂. Figure 6 shows representative radio-HPLC chromatograms of ^99mTc-Galacto-RGD₂ in saline before being injected into the 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-Galacto-RGD₂. More than 90% of ^99mTc-Galacto-RGD₂ remained intact in the feces sample at 2 h p.i. Its metabolic stability was better than that of ^99mTc-3P–RGD₂, only 70% of which remained intact in the feces at 2 h p.i.³¹

Figure 6.

Typical radio-HPLC chromatograms for ^99mTc-Galacto-RGD₂ 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. Small peaks after the main peak at ~13 min were at the noise level due to the extremely small amount of radioactivity isolated from feces sample for HPLC analysis.

DISCUSSION

In this study, we prepared a novel RGD peptide dimer, Galacto-RGD₂, which contains a pair of SAA and 1,2,3-triazole groups. A similar approach was used to develop other integrin α_vβ₃-targeted radiotracers,^52,53 such as ¹⁸F-RGD-K5, in which 1,2,3-triazole is formed via click reaction to attach the ¹⁸F-containing fragment to Galacto-RGD.⁵² In Galacto-RGD₂, the SAA and 1,2,3-triazole moieties are parts of a linker with two extra PEG₂ groups and a glutamic acid residue. Boc-Glutamic acid bis-propargyl amide was a key intermediate for synthesis of Galacto-RGD₂. It may become an useful building block for click-type of dimerization/multimerization reactions of peptides and other organic molecules via the Cu(I)-catalyzed Huisgen cycloaddition. Click chemistry has proven valuable in development radiopharmaceuticals,^52,54–60 including radiolabeled multimeric cyclic RGD peptides.^55–60

The integrin α_vβ₃ binding affinity of HYNIC-Galacto-RGD₂ (IC₅₀ = 20±2 nM) was slightly higher than that of HYNIC-3P–RGD₂ (IC₅₀ = 32±1 nM), likely due to the rigidity of SAA and 1,2,3-triazole moieties in Galacto-RGD₂ as compared to more flexible PEG₄ groups in 3P–RGD_2. It is important to note that IC₅₀ values depends on the radioligand (¹²⁵I-c(RGDyK) vs. ¹²⁵I-echistatin) and tumor cell lines (U87MG vs. MDA-MB-435) used in the competitive assay. Caution should be taken when comparing IC₅₀ values of cyclic RGD peptides with those reported in the literature.

^99mTc-Galacto-RGD₂ and ^99mTc-3P–RGD₂ shared a similar tumor uptake (Figure 4A) despite of slightly higher integrin α_vβ₃ binding affinity of HYNIC-Galacto-RGD₂ than that of HYNIC-3P–RGD₂. This is most likely related to the fact that both ^99mTc-Galacto-RGD₂ and ^99mTc-3P–RGD₂ are bivalent in binding to integrin α_vβ₃. As a result, the small difference in their integrin α_vβ₃ binding affinity was not reflected in tumor uptake of their ^99mTc radiotracers. This explanation is also supported by much lower tumor uptake of [^99mTc(HYNIC-RGD₂)(tricine)(TPPTS)] (^99mTc-RGD₂), which is only monovalent in binding to integrin α_vβ₃ despite of the presence of two cyclic RGD moieties in RGD₂.³¹ Bivalency remains the key factor that contributes to the tumor uptake of integrin α_vβ₃-targeted radiotracers.¹⁸

It must be noted that bivalency is largely dependent on integrin α_vβ₃ receptor density on a specific tissue. In the tumors like xenografted U87MG gliomas, the integrin α_vβ₃ density is high, and the distance between two neighboring integrin α_vβ₃ sites is relatively short. Therefore, it is possible for ^99mTc-Galacto-RGD₂ or ^99mTc-3P–RGD₂ to be bivalent in binding to integrin α_vβ₃. In most normal organs, however, the integrin α_vβ₃ density is relatively low and the distance between two integrin α_vβ₃ sites is expected to be much longer. In this situation, ^99mTc-Galacto-RGD₂ and ^99mTc-3P–RGD₂ may not be bivalent in binding to integrin α_vβ₃ expressed on normal tissues.

The major difference between ^99mTc-Galacto-RGD₂ and ^99mTc-3P–RGD₂ is that two PEG₄ groups between the two RGD moieties in 3P–RGD₂ are replaced with a pair of SAA, 1,2,3-triazole and PEG₂ moieties. This change resulted in a faster radiotracer washout from tumors (Table 1) and normal organs, most likely due to the improved hydrophilicity. Reduction of radiotracer uptake in the intestines, lungs and spleen (Figure 4A) is important for diagnosis of tumors in chest and abdominal regions. The integrin α_vβ₃ specificity of ^99mTc-Galacto-RGD₂ was demonstrated in the blocking study (Figure 4B). ^99mTc-Galacto-RGD₂ was excellent for imaging the xenografted U87MG tumors (Figure 5A). Its high glioma uptake was completely in agreement with the high integrin α_vβ₃ expression on both U87MG glioma cells (Figure 5B) and tumor neovasculature (Figure 5C). ^99mTc-Galacto-RGD₂ also had better metabolic stability than ^99mTc-3P–RGD₂ during hepatobiliary excretion (Figure 6). All these factors make us to believe that ^99mTc-Galacto-RGD₂ has advantages over ^99mTc-3P–RGD₂ for imaging tumors and related metastases in the chest and abdominal regions.

In our previous study, it has been clearly demonstrated that many normal organs (particularly intestine, liver, lung, kidneys and spleen) are integrin α_vβ₃–positive.³⁸ This is consistent with the reduced uptake of ^99mTc-Galacto-RGD₂ in these organs (Figure 4B) in the presence of co-injected excess RGD₂. When part of the radiotracer uptake is blocked in integrin α_vβ₃–positive organs, it is not surprising to see elevated blood radioactivity accumulation. However, it doesn’t mean that the radioactivity accumulation in these organs is all integrin α_vβ₃–oriented. For example, the uptake of ^99mTc-Galacto-RGD₂ in liver and lung is reduced only by 50%, but its intestine uptake was reduced by ~80% of its origin value. This conclusion is further supported by comparing 60-min biodistribution data of ¹¹¹In(DOTA-3P–RGD₂) and ¹¹¹In(DOTA-3P–RGK₂), which shows very low integrin α_vβ₃ binding affinity (IC₅₀ = 715.8 ± 45.1 nM), but still has significant uptake in the intestine, lung, kidneys and spleen.³⁹

CONCLUSION

In this study, we evaluated ^99mTc-Galacto-RGD₂ as a new SPECT radiotracer for imaging the xenografted U87MG glioma tumors. We found that ^99mTc-Galacto-RGD₂ could be prepared in high specific activity, and was able to maintain its solution stability for >24 h post-labeling. Because of its lower uptake in intestines, lungs and spleen, ^99mTc-Galacto-RGD₂ has advantages over ^99mTc-3P–RGD₂ for imaging tumors in the chest and abdominal regions. ^99mTc-Galacto-RGD₂ also has a very high metabolic stability. On the basis of results from this and our previous studies,^31–44 we strongly believe that ^99mTc-Galacto-RGD₂ is an excellent radiotracer for imaging integrin α_vβ₃-positive tumors and related metastases.

Figure 3.

Radio-HPLC chromatograms of ^99mTc-Galacto-RGD₂ in the kit matrix at 1 and 24 h post-labeling. The radiometric peak at ~13.5 min was from ^99mTc-Galacto-RGD₂. Its radiochemical purity was always >95% without any chromatographic purification.