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. Author manuscript; available in PMC: 2010 Dec 21.
Published in final edited form as: Bioconjug Chem. 2009 Jul 15;20(8):1559–1568. doi: 10.1021/bc9001739

2-Mercaptoacetylglycylglycyl (MAG2) as a Bifunctional Chelator for 99mTc-Labeling of Cyclic RGD Dimers: Effect of Technetium Chelate on Tumor Uptake and Pharmacokinetics

Jiyun Shi 1,2, Young-Seung Kim 1, Sudipta Chakraborty 1, Bing Jia 2, Fan Wang 2, Shuang Liu 1,*
PMCID: PMC2888811  NIHMSID: NIHMS132874  PMID: 19603780

Abstract

This report describes the synthesis of MAG2-PEG4-E[c(RGDfK)]2 (MAG2-P-RGD2: MAG2 = S-benzoylmercaptoacetylglycylglycyl; PEG4 = 15-amino-4,7,10,13-tetraoxapentadecanoic acid) and MAG2-PEG4-E[PEG4-c(RGDfK)]2 (MAG2-3P-RGD2), and the evaluation of 99mTcO(MAG2-P-RGD2) and 99mTcO(MAG2-3P-RGD2) as new radiotracers for tumor imaging in the athymic nude mice bearing U87MG human glioma xenografts. We found that MAG2 is such an efficient bifunctional chelating agent that 99mTcO(MAG2-P-RGD2) and 99mTcO(MAG2-3P-RGD2) could be prepared in high yield (>90%) with high specific activity (∼5 Ci/μmol) using a kit formulation. 99mTcO(MAG2-P-RGD2) and 99mTcO(MAG2-3P-RGD2) have very high solution stability in the kit matrix. Biodistribution data in athymic nude mice bearing U87MG human glioma xenografts indicated that replacing the highly charged [99mTc(HYNIC)(tricine)(TPPTS)] (6-hydrazinonicotinyl and TPPTS = trisodium triphenylphosphine-3,3′,3″-trisulfonate) with smaller 99mTcO(MAG2) resulted in a significant increase in the radiotracer uptake in the tumor and normal organs most likely due to the higher lipophilicity of 99mTcO(MAG2-3P-RGD2) (log P = -3.15 ± 0.10) than that for [99mTc(HYNIC-3P-RGD2)(tricine)(TPPTS)] (99mTc-3P-RGD2: log P = -3.96 ± 0.05). Even though 99mTcO(MAG2-3P-RGD2) has better tumor uptake (15.48 ± 2.49 %ID/g at 60 min postinjection (p.i.)) than 99mTc-3P-RGD2 (9.15 ± 2.13 %ID/g at 60 min p.i.), its tumor-to-background (T/B) ratios (tumor/blood = 13.02 ± 6.12; tumor/liver = 4.07 ± 0.95; tumor/lung = 2.97 ± 0.64; and tumor/muscle = 8.04 ± 0.43) are not as good as those of 99mTc-3P-RGD2 (tumor/blood = 36.0 ± 11.5; tumor/liver = 5.14 ± 1.46; tumor/lung = 4.36 ± 0.54; and tumor/muscle = 13.70 ± 2.21) at 60 min p.i. On the basis of these results, we believe that 99mTc-3P-RGD2 remains a better radiotracer because of its higher T/B ratios.

Keywords: integrin αvβ3, 99mTc -labeled cyclic RGD peptides, tumor imaging

Introduction

Radiolabeled cyclic RGD (Arg-Gly-Asp) peptides represent a new class of radiotracers that target the integrin αvβ3 overexpressed on both tumor cells and endothelial cells of neovasculature during tumor growth, invasion and metastasis (1-12). Many radiolabeled cyclic RGD peptides have been evaluated for imaging integrin αvβ3-positive tumors by single photon emission computed tomography (SPECT) or positron emission tomography (PET) over the last several years, (13-49). Among the RGD peptide radiotracers evaluated in different tumor-bearing animal models, [18F]-AH111585, the core peptide sequence discovered from a phage display library (such as ACDRGDCFCG) (50, 51), and [18F]Galacto-RGD (2-[18F]fluoropropanamide c(RGDfK(SAA); SAA = 7-amino-L-glyero-L-galacto-2,6-anhydro-7-deoxyheptanamide) (52-54) are currently under clinical investigations for noninvasive visualization of integrin αvβ3 expression in cancer patients. The imaging studies show that the 18F-labeled RGD peptides are able to target integrin αvβ3–positive tumors (51-54). However, the low tumor uptake, high cost and lack of preparation modules for 18F-labeled cyclic RGD peptides impose a significant challenges their continued clinical applications.

To improve integrin αvβ3 binding affinity and radiotracer tumor uptake, multimeric cyclic RGD peptides, such as E[c(RGDfK)]2 and E{E[c(RGDfK)]2}2, were used as targeting biomolecules to carry radionuclide (e.g. 99mTc, 18F, 64Cu, and 111In) to the integrin αvβ3 on tumor cells and endothelial cells of the tumor neovasculature (20-22, 30-35,37-49). The results from in vitro assays, ex-vivo biodistribution and in vivo imaging studies clearly demonstrate that the radiolabeled (99mTc, 18F, 64Cu, and 111In) multimeric cyclic RGD peptides, such as E{E[c(RGDxK)]2}2 and E[c(RGDxK)]2 (x = f and y), have better tumor targeting capability as evidenced by their higher integrin αvβ3 binding affinity, better tumor uptake with longer tumor retention times than their monomeric counterparts (20-22, 30-35,37-49). However, the uptake of radiolabeled (99mTc, 18F, 64Cu and 111In) multimeric cyclic RGD peptides in the kidneys and liver is also increased as the peptide multiplicity increases.

Recently, we reported the evaluation of the complex [99mTc(HYNIC-3P-RGD2)(tricine)(TPPTS)] (Figure 1: 99mTc-3P-RGD2; HYNIC = 6-hydrazinonicotinyl; TPPTS = trisodium triphenylphosphine-3,3′,3″-trisulfonate; 3P-RGD2 = PEG4-E[PEG4-c(RGDfK)]2 and PEG4 = 15-amino-4,7,10,13-tetraoxapentadecanoic acid) as radiotracers for imaging integrin αvβ3 expression in athymic nude mice bearing U87MG glioma and MDA-MB-435 breast cancer xenografts (55). The PEG4 linkers are used to increase the distance between two cyclic RGD motifs from 6 bonds (excluding side arms of K-residues) in E[c(RGDfK)]2 to 38 bonds in 3P-RGD2 in so that they are able to achieve the simultaneous integrin αvβ3 binding in a bivalent fashion, and to improve radiotracer excretion kinetics from noncancerous organs. Results from the αvβ3 integrin binding assay show that the addition of PEG4 linkers between two cyclic RGD motifs makes it possible for 3P-RGD2 to become bivalent in binding to the integrin αvβ3. The results from biodistribution studies clearly demonstrate that PEG4 and G3 linkers are useful for improving the tumor uptake and clearance kinetics of 99mTc radiotracers from noncancerous organs (55, 56). Similar results were also obtained for [99mTc(HYNIC-3G-RGD2)(tricine)(TPPTS)] (99mTc-3G-RGD2: 3G-RGD2 = G3-E[G3-c(RGDfK)]2 and G3 = Gly-Gly-Gly) (56), 64Cu(DOTA-3P-RGD2) (DOTA = 1,4,7,10-tetraazacyclo-dodecane-1,4,7,10-tetracetic acid) and 64Cu(DOTA-3G-RGD2) (57).

Figure 1.

Figure 1

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Structures of bivalent cyclic RGD dimers (P-RGD2 and 3P-RGD2) and their 99mTc complexes: [99mTc(HYNIC-3P-RGD2)(tricine)(TPPTS)] (99mTc-3P-RGD2), 99mTcO(MAG2-P-RGD2) and 99mTcO(MAG2-3P-RGD2).

As a continuation of our interest in radiolabeled cyclic RGD peptides as radiotracers for tumor imaging (41-44, 55, 56), we prepared two novel cyclic RGD conjugates, MAG2-PEG4-E[c(RGDfK)]2 (MAG2-P-RGD2: MAG2 = S-benzoylmercaptoacetylglycylglycyl) and MAG2-3PEG4-E[PEG4-c(RGDfK)]2 (MAG2-3P-RGD2), and their 99mTc complexes (Figure 1: 99mTcO(MAG2-P-RGD2) and 99mTcO(MAG2-3P-RGD2). MAG2 is of our particular interest as the bifunctional chelator (BFC) because it forms the 99mTcO(MAG2) chelate (molecular weight (MW) ∼300 Daltons) that is much smaller than the bulky and highly charged [99mTc(HYNIC)(tricine)(TPPTS)] (MW ∼ 900 Daltons). The integrin αvβ3 binding affinities of MAG2-P-RGD2 and MAG2-3P-RGD2 were determined using a displacement assay against 125I-c(RGDyK) bound to U87MG glioma cells. Biodistribution characteristics of 99mTcO(MAG2-P-RGD2) and 99mTcO(MAG2-3P-RGD2) were evaluated in the athymic nude mice bearing U87MG glioma xenografts. The main objective of this study is to demonstrate the usefulness of MAG2 as a BFC for 99mTc-labeling of small biomolecules, and to assess the impact of 99mTc chelates (99mTcO(MAG2) vs. [99mTc(HYNIC)(tricine)(TPPTS)]) on both tumor uptake and tumor-to-background (T/B) ratios.

Experimental Section

Materials

Chemicals were purchased from Sigma-Aldrich (St. Louis, MO), and were used without further purification. Cyclic RGD peptides, PEG4-E[PEG4-c(RGDfK)]2 (3P-RGD2) and PEG4-E[PEG4-c(RGKfD)]2 (3P-RGK2: a scrambled nonsense peptide), were custom-made by the Peptides International, Inc. (Louisville, KY). MAG2 (S-benzoylmercaptoacetylglycylglycine) and PEG4-E[c(RGDfK)]2 (P-RGD2) were prepared according to the literature methods (43, 58). Na99mTcO4 was obtained from a commercial DuPont Pharma 99Mo/99mTc generator (North Billerica, MA). The ESI (electrospray ionization) mass spectral data were collected on a Finnigan LCQ classic mass spectrometer, School of Pharmacy, Purdue University.

HPLC Methods

HPLC Method 1 used a LabAlliance HPLC system equipped with a UV/vis detector (λ=254 nm) and Zorbax C18 semi-prep column (9.4 nm × 250 mm, 100 Å pore size; Agilent Technologies, Santa Clara, CA). The flow rate was 2.5 mL/min with the mobile phase starting from 90% A (25 mM NH4OAc, pH = 6.8) and 10% B (acetonitrile) at 0 min, followed by a gradient mobile phase going from 85% A and 15% B at 5 min to 65% A and 35% B at 30 min. The radio-HPLC method (Method 2) 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 with 90% A (25 mM NH4OAc, pH = 6.8) and 10% B (acetonitrile) at 0 – 2 min, followed by a gradient mobile phase going from 10% B at 2 min to 15% B at 5 min and to 20% B at 20 min. The radio-ITLC method used GelmanSciences silica-gel paper strips and a 1:1 mixture of acetone and saline as eluant. By this method, 99mTc-labeled cyclic RGD peptides migrate to the solvent front while 99mTcO4- and [99mTc]colloid remain at the origin.

MAG2-OSu

To a solution of MAG2 (310 mg, 1 mmol) and N-hydroxysuccinimide (130 mg, 1.1 mmol) in DMF (5 mL) was added dicyclcohexylcarbodiimide (DCC: 230 mg, 1.1 mmol). The mixture was stirred at room temperature for 24 h. After addition of 0.3 mL acetic acid, the resulting mixture was stirred at room temperature for another 5 h. The precipitate was filtered, and discarded. The filtrate was evaporated to dryness on a rotary evaporator. The residue was dissolved in dichloromethane (10 mL). After filtration, the filtrate was concentrated to ∼2 mL. The solution was added dropwise into diethyl ether (20 mL) to give an off-white precipitate. The solid product was collected, washed with diethyl ether, and dried under vacuum. The yield was 340 mg (83%). 1H NMR (CDCl6, chemical shifts in ppm relative to TMS): 2.81 (s, 4H, COCH2CH2CO); 3.78 (s, 2H, SCH2CO); 4.03 (d, 2H, CH2CO); 4.38 (d, 2H, CH2CO); 7.13 (dt, 2H, aromatic); 7.47 (t, 2H, aromatic); 7.61 (t, H, aromatic); and 7.96 (d, 2H, NHCO). ESI-MS: m/z = 408.50 for [M+H]+ (408 calcd. for [C17H18N3O7S]+).

MAG2-P-RGD2

MAG2-OSu (5.8 mg, 14.2 μmol) and P-RGD2 (5.4 mg, 3.45 μmol) were dissolved in DMF (2 mL). To the mixture above was added diisopropylethylamine (DIEA, 2 drops). The solution was stirred at room temperature for 2 h. After addition of water (2 mL), the pH was adjusted to 3.0 – 4.0. The product was separated from the reaction mixture by HPLC (Method 1). Fractions at 17.5 min were collected. Lyophilization of combined collections afforded the crude product MAG2-P-RGD2 (∼75% purity by HPLC), which was then further purified by HPLC (Method 2). The yield was 2.7 mg (42%). ESI-MS: m/z = 1858.43 for (1857 calcd. For [C84H121N22O25S]+).

MAG2-3P-RGD2

MAG2-3P-RGD2 was prepared in a similar fashion as that for MAG2-P-RGD2 using MAG2-OSu (4 mg, 9.7 μmol) and 3P-RGD2 (5 mg, 2.43 μmol). The product was purified by HPLC (Method 1). Fractions at ∼16 min were collected. Lyophilization of the combined collections afforded the crude product MAG2-3P-RGD2 (∼77% purity by HPLC), which was further purified by HPLC (Method 2). Lyophilization of the combined collections at 20 min afforded MAG2-3P-RGD2 with >95% HPLC purity. The yield was 2.0 mg (35%). ESI-MS: m/z = 2351.79 for (2351 calcd. for [C105H163N24O35S]+).

MAG2-PEG4-E[PEG4-c(RGKfD)]2 (MAG2-3P-RGK2)

MAG2-3P-RGK2 was prepared according to the same procedure used for MAG2-PEG4-dimer using MAG2-OSu (12.5 mg, 40 μmol) and 3P-RGK2 (15 mg, 7.29 μmol). The product was separated from the reaction mixture by HPLC (Method 1). Fractions at 16 min were collected. Lyophilization of the combined collections afforded the crude product MAG2-3P-RGK2 (∼70% purity by HPLC), which was then further purified by HPLC (Method 2). Lyophilization of the combined collections at 19 min afforded MAG2-3P-RGK2 with >95% HPLC purity. The yield was 6.2 mg (36%). ESI-MS: m/z = 2351.76 for (2351 calcd. for [C105H163N24O35S]+).

99mTc-Labeling

To a lyophilized vial containing 2.28 mg of NaH2PO4, 11.5 mg of Na2HPO4, 50 mg of α-D-glucoheptonic acid, 50 μg of SnCl2·2H2O, and 25 μg of the MAG2-conjugate (MAG2-P-RGD2, MAG2-3P-RGD2 or MAG2-3P-RGD2) was added 1.0 mL of Na99mTcO4 solution (10 – 50 mCi/mL). The vial was heated at 100 °C for 20 – 25 min in a lead-shielded water bath. After heating, the vial was placed back into the lead pig, and allowed to stand at room temperature for ∼10 min. A sample of the resulting solution was analyzed by radio-HPLC (Method 2) and radio-ITLC. The radiochemical purity (RCP) was >95% for all three radiotracers, 99mTcO(MAG2-P-RGD2), 99mTcO(MAG2-3P-RGD2) and 99mTcO(MAG2-3P-RGK2), with less than 0.5% of [99mTc]colloid.

Dose Preparation

For the ex-vivo biodistribution studies, the 99mTc radiotracers were first prepared, and were then purified by HPLC (Method 2). Volatiles in the HPLC mobile phases were removed under vacuum. The dose solution was prepared by dissolving the purified radiotracer in saline to a concentration of 10 – 25 μCi/mL. In the blocking experiment, E[c(RGDfK)]2 was dissolved in the dose solution to ∼3.5 mg/mL. For imaging studies, 99mTcO(MAG2-3P-RGD2) was prepared in high yield (RCP > 95%). Doses were made by dissolving the reconstituted kit solution to a concentration of 5 mCi/mL. The solution was filtered with a 0.20 μ Millex-LG filter to remove any particle or precipitate. Each tumor-bearing mouse was injected with ∼0.1 mL of the filtered dose solution.

Determination of Log P Values

Log P values of were determined using the following procedure: the 99mTc radiotracer was purified by HPLC. Volatiles were removed completely under vacuum. The residue was dissolved in a equal volume (3 mL:3 mL) mixture of n-octanol and 25 mM phosphate buffer (pH = 7.4). After stirring vigorously for ∼20 min, the mixture was centrifuged at a speed of 8,000 rpm for 5 min. Samples (in triplets) from n-octanol and aqueous layers were counted in a Perkin Elmer Wizard – 1480 γ-counter (Shelton, CT). The log P value was measured three different times and reported as an average of three independent measurements plus the standard deviation.

In Vitro Whole-Cell Integrin αvβ3 Binding Assay

The in vitro integrin binding affinity of cyclic RGD peptides were assessed by a displacement assay using 125I-c(RGDyK) as the integrin αvβ3-specific radioligand. Experiments were performed on U87MG human glioma cells by slight modification of a method previously described (28, 30). Briefly, U87MG glioma cells were grown in Gibco's Dulbecco's medium supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin and 100 μg/ml streptomycin (Invitrogen Co, Carlsbad, CA), at 37 °C in humidified atmosphere containing 5% CO2. Filter multiscreen DV plates were seeded with 105 cells in binding buffer and incubated with 125I-c(RGDyK) in the presence of increasing concentrations of cyclic RGD peptides. After removing the unlabeled c(RGDyK), hydrophilic PVDF filters were collected and the radioactivity was determined using a gamma counter (Packard, Meriden, CT). The IC50 values were calculated by fitting the data by nonlinear regression using GraphPad PrismTM (GraphPad Software, Inc., San Diego, CA), and reported as an average plus the standard deviation. Comparison between two radiotracers was made using the two-way ANOVA test (GraphPad Prim 5.0, San Diego, CA). The level of significance was set at p < 0.05.

Animal Model

Biodistribution studies were performed using the athymic nude mice bearing U87MG human glioma xenografts in compliance the NIH animal experiment guidelines (Principles of Laboratory Animal Care, NIH Publication No. 86-23, revised 1985). The protocol was approved by Purdue University Animal Care and Use Committee (PACUC). The U87MG human glioma cells were grown at 37 °C in Minimal Essential Medium (Alpha Modification) containing 3.7 g of sodium bicarbonate/L, 10% fetal bovine serum v/v, and 5% Penicillin Streptomycin (GIBCO, Grand Island, NY) in a humidified atmosphere of 5% carbon dioxide. Female athymic nu/nu mice were purchased from NCI at 4 – 5 weeks of age. The mice were implanted subcutaneously with ∼8×106 U87MG human glioma cells into the upper left flanks. All procedures will be performed in a laminar flow cabinet using aseptic technique. Two to three weeks after inoculation, the tumor size was 0.1 – 0.4 g, and animals were used for biodistribution and imaging studies.

Biodistribution Protocol

Fifteen tumor-bearing mice (20 – 25 g) were randomly divided into four groups. Each animal was administered with 99mTc radiotracer (∼2.5 μCi in 0.1 mL saline) via tail vein. Five animals were euthanized by sodium pentobarbital overdose (100 – 200 mg/kg) at 30, 60 and 120 min postinjection (p.i.). Blood samples were withdrawn from the heart. The tumor and normal organs (brain, eyes, heart, intestine, kidneys, liver, lungs, muscle and spleen) were excised, washed with saline, dried with absorbent tissue, weighed, and counted on a γ-counter (Perkin Elmer Wizard – 1480). The organ uptake was calculated as the percentage of injected dose per gram of organ tissue (%ID/g). For the blocking experiment to demonstrate the radiotracer integrin αvβ3-specificity, each animal was administered with ∼2 μCi of 99mTcO(MAG2-3P-RGD2) along with ∼350 μg of E[c(RGDfK)]2 (∼14 mg/kg). At 1 h p.i., five animals were sacrificed by sodium pentobarbital overdose (100 mg/kg) for organ biodistribution. The organ uptake (%ID/g) was compared to that obtained in the absence of excess E[c(RGDfK)]2 at the same time point. For the experiment to demonstrate the radiotracer RGD-specific, each animal was administered with ∼2 μCi of 99mTcO(MAG2-3P-RGD2) along with ∼350 μg of E[c(RGDfK)]2 (∼14 mg/kg). At 1 h p.i., five animals were sacrificed by sodium pentobarbital overdose (100 mg/kg) for organ biodistribution. The organ uptake (%ID/g) was compared to that obtained in the absence of excess E[c(RGDfK)]2 at the same time point.

Scintigraphic Imaging

Three athymic nude mice bearing U87MG glioma xenografts were used for planar imaging studies. Each glioma-bearing mouse was intravenously administered with ∼500 μCi of 99mTcO(MAG2-3P-RGD2) via tail vein. The animals were anesthetized with intramuscular injection of ketamine (80 mg/kg) and xylazine (19 mg/kg). The animal was placed prone on a single head mini γ-camera (Diagnostic Services Inc., NJ) equipped with a parallel-hole, low-energy, and high-resolution collimator. Static images were acquired at 15, 30, 60 and 120 min p.i. and were stored digitally in a 128×128 matrix. The acquisition count limits were set at 500 K. After imaging, the animals were euthanized by sodium pentobarbital overdose (100 – 200 mg/kg).

Metabolism

Three glioma-bearing mice were used for the in vivo metabolic stability study of 99mTcO(MAG2-3P-RGD2), which was intravenously administered at the dose of ∼100 μCi per mouse via tail vein. At 120 min p.i., the urine samples were collected, and were mixed with equal volume of 20% acetonitrile aqueous solution. The mixture was centrifuged at 8,000 rpm for 5 min. The supernatant was collected and filtered through a 0.20 μ Millex-LG filter unit. The feces samples were collected at 120 min p.i. and suspended in a mixture of 20% acetonitrile aqueous solution (2 mL). The resulting mixture was vortexed for 10 min. After centrifuging at 8,000 rpm for 5 min, the supernatant was collected and passed through a 0.20 μ Millex-LG filter unit to remove any particles or precipitate. Both the urine and feces samples were analyzed by radio-HPLC (Method 2). The tumor, kidney, and liver tissues were harvested at 120 min p.i. counted in a Perkin Elmer Wizard – 1480 γ-counter (Shelton, CT) for total radioactivity, and was then homogenized. The homogenate was mixed with 2 mL of 20% acetonitrile aqueous solution. After centrifuging at 8,000 rpm for 5 min, the supernatant was collected and counted on a γ-counter to determine the percentage of radioactivity recovery in each organ. After filtration through a 0.20 μm Millex-LG filter unit to remove foreign particles or precipitate, the filtrate was then analyzed by radio-HPLC (Method 2).

Data and Statistical Analysis

The biodistribution data and target-to-background (T/B) ratios are reported as an average plus the standard variation based on results from four tumor-bearing mice at each time point. Comparison between two different 99mTc radiotracers was made using the two-way ANOVA test (GraphPad Prim 5.0, San Diego, CA). The level of significance was set at p < 0.05.

Results

MAG2 Conjugate Synthesis

Synthesis of new cyclic RGD peptide conjugates (MAG2-P-RGD2 and MAG2-3P-RGD2) was straightforward. They were prepared by conjugation of P-RGD2 and 3P-RGD2, respectively, with excess MAG2-NHS in DMF in the presence of a base, such as DIEA. MAG2-3P-RGK2 contains two c(RGKfD) motifs instead of c(RGDfK) motifs in MAG2-3P-RGD2, and was prepared as a “negative control” to demonstrate the RGD-specificity of 99mTcO(MAG2-3P-RGD2). All new peptide conjugates were purified twice by HPLC (Method 1), and characterized by ESI-MS. Their HPLC purity was >95% before being used for 99mTc-labeling and determination of their integrin αvβ3 binding affinity.

Integrin αvβ3 Binding Affinity

The integrin αvβ3 binding affinity of c(RGDfK), MAG2-3P-RGD2 and MAG2-3P-RGD2 and MAG2-3P-RGK2 were determined by a competitive displacement assay. Their IC50 values were obtained from curve fitting from Figure 2, and were calculated to be 46.6 ± 4.5, 8.6 ± 2.8, 3.9 ± 0.4 and 711 ± 128 nM, respectively.

Figure 2.

Figure 2

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The in vitro inhibition curve of 125I-c(RGDyK) bound to integrin αvβ3 on U87MG human glioma cells by c(RGDyK), MAG2-P-RGD2, MAG2-3P-RGD2 and MAG2-3P-RGK2. Their IC50 values were calculated to be 46.6 ± 4.5, 8.6 ± 2.8, 3.9 ± 0.4 and 711 ± 128 nM, respectively.

Radiochemistry

99mTcO(MAG2-P-RGD2) and 99mTcO(MAG2-3P-RGD2) were prepared by reacting MAG2-P-RGD2 and MAG2-3P-RGD2, respectively, with 99mTcO4- at pH ∼8.5 in the presence of excess D-glucoheptonic acid (∼50 mg per vial) and stannous chloride. 99mTc-labeling was accomplished by heating the reaction mixture at 100 °C for 15 – 20 min. D-glucoheptonic acid was used to stabilize SnCl2 and the [99mTcO]3+ core, and to prevent formation of [99mTc]colloid during radiolabeling. Their radiochemical purity was >90% with <0.5% of [99mTc]colloid. In general, 20 – 25 μg MAG2-3P-RGD2 was sufficient for successful radiolabeling of 50 mCi of 99mTcO4-. The specific activity for 99mTcO(MAG2-3P-RGD2) was ∼2.0 mCi/μg (∼5 Ci/μmol). 99mTcO(MAG2-3P-RGD2) was stable in the kit matrix for >6 h. All 99mTc radiotracers were analyzed by the same HPLC method, and their retention times were listed in Table 1. The log P values for 99mTcO(MAG2-P-RGD2), 99mTcO(MAG2-3P-RGD2) and 99mTcO(MAG2-3P-RGK2) were determined to be -3.30 ± 0.13, -3.19 ± 0.10 and -2.40 ± 0.14, respectively.

Table 1.

HPLC retention time and log P values for 99mTc-labeled cyclic RGD peptides.

Compound RCP (%) RT (min) Log P
99mTcO(MAG2-P-RGD2) > 95 17.0 - 3.30 ± 0.13
99mTcO(MAG2-3P-RGD2) > 90 16.9 -3.19 ± 0.15
99mTcO(MAG2-3P-RGK2) > 90 17.3 -2.40 ± 0.14
99mTc-3P-RGD2 > 90 16.9 -4.35 ± 0.10*
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*

The log P value of −3.96 ± 0.05 was reported in our previous communication (55). These two values seem to be statistically significant; but they are within the experimental error of the assay.

Biodistribution Characteristics

The biodistribution data for 99mTcO(MAG2-P-RGD2) and 99mTcO(MAG2-3P-RGD2) were listed in Tables 2 and 3, respectively. 99mTcO(MAG2-P-RGD2) had the tumor uptake of 10.41 ± 2.07 and 11.56 ± 1.41 %ID/g at 30 and 60 min, respectively, but it decreased to 5.66 ± 0.50 %ID/g at 120 min p.i. It also had a high kidney uptake with a fast clearance (19.01 ± 1.48 and 4.02 ± 0.81 % ID/g at 30 and 120 min p.i., respectively). The liver uptake of 99mTcO(MAG2-P-RGD2) was 4.84 ± 0.87, 3.11 ± 0.30 and 1.43 ± 0.20 %ID/g while its tumor/liver ratios were 2.34 ± 0.58, 3.78 ± 0.27 and 3.99 ± 0.52 at 30, 60 and 120 min p.i., respectively. 99mTcO(MAG2-3P-RGD2) also had a high tumor uptake (16.78 ± 5.46, 15.48 ± 2.49 and 13.60 ± 2.30 %ID/g at 30, 60 and 120 min p.i., respectively). Its kidney uptake was 18.68 ± 2.10, 13.38 ± 2.07 and 6.43 ± 0.89 % ID/g at 30, 60 and 120 min p.i. respectively. Its liver uptake was 4.97 ± 0.68 %ID/g at 30 min p.i. and 2.38 ± 0.17 %ID/g at 120 min p.i., and its tumor/liver ratio increased steadily from 3.39 ± 1.03 at 30 min p.i. to 5.71 ± 0.82 at 120 min p.i.

Table 2.

Biodistribution data of 99mTcO(MAG2-P-RGD2) in athymic nude mice (n = 5) bearing U87MG human glioma xenografts.

30 min 60 min 120 min
Blood 1.50 ± 0.64 0.85 ± 0.42 0.11 ± 0.02
Brain 0.45 ± 0.29 0.54 ± 0.55 0.09 ± 0.01
Eyes 2.21 ± 0.80 2.14 ± 0.55 0.94 ± 0.14
Heart 2.51 ± 1.13 1.60 ± 0.54 0.71 ± 0.10
Intestine 9.39 ± 2.92 5.25 ± 1.08 3.27 ± 1.14
Kidney 16.61 ± 5.52 9.95 ± 3.90 3.92 ± 0.60
Liver 4.25 ± 1.53 2.75 ± 0.76 1.47 ± 0.15
Lungs 5.41 ± 1.62 2.65 ± 0.57 2.28 ± 0.72
Muscle 3.33 ± 0.77 2.53 ± 1.10 1.12 ± 0.26
Spleen 3.52 ± 1.13 3.04 ± 1.09 1.25 ± 0.12
U87MG 9.63 ± 1.39 11.95 ± 1.90 5.90 ± 0.35
Tumor/Blood 8.53 ± 3.72 12.58 ± 2.60 46.62 ± 4.17
Tumor /Kidney 0.59 ± 0.08 1.15 ± 0.05 1.49 ± 0.28
Tumor /Liver 2.54 ± 0.68 2.98 ± 0.29 3.69 ± 0.58
Tumor /Lungs 1.95 ± 0.40 3.25 ± 1.13 2.93 ± 0.68
Tumor /Muscle 2.98 ± 0.35 4.20 ± 0.75 4.79 ± 0.54
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Table 3.

Biodistribution data of 99mTcO(MAG2-3P-RGD2) in athymic nude mice (n = 5) bearing U87MG human glioma xenografts.

%ID/gram 30 min 60 min 120 min
Blood 1.62 ± 0.69 1.16 ± 0.78 0.42 ± 0.29
Brain 0.26 ± 0.07 0.21 ± 0.06 0.17 ± 0.07
Eyes 2.47 ± 1.34 1.40 ± 0.65 1.48 ± 0.77
Heart 2.40 ± 0.97 1.79 ± 0.45 1.05 ± 0.18
Intestine 10.99 ± 1.95 9.26 ± 1.50 6.58 ± 1.62
Kidney 16.35 ± 5.53 11.79 ± 3.98 6.07 ± 1.12
Liver 4.35 ± 1.51 3.45 ± 0.86 2.30 ± 0.23
Lungs 6.15 ± 1.93 4.17 ± 1.35 2.78 ± 0.49
Muscle 2.17 ± 0.35 1.82 ± 0.83 0.97 ± 0.24
Spleen 3.41 ± 1.12 3.02 ± 0.56 1.98 ± 0.14
U87MG 15.49 ± 5.55 15.36 ± 2.17 14.02 ± 2.20
Tumor/Blood 9.15 ± 2.51 13.52 ± 4.57 31.54 ± 10.23
Tumor /Kidney 0.98 ± 0.30 1.25 ± 0.36 2.01 ± 0.12
Tumor /Liver 3.49 ± 1.13 4.25 ± 0.88 5.81 ± 0.65
Tumor /Lung 2.51 ± 0.62 3.17 ± 0.60 4.95 ± 1.00
Tumor /Muscle 6.60 ± 1.25 8.34 ± 2.34 12.19 ± 1.07
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Effects of Technetium Chelate

To assess the impact of 99mTc chelates (99mTcO(MAG2) vs. [99mTc(HYNIC)(tricine)(TPPTS)]) on the radiotracer tumor uptake and T/B ratios, we also obtained the 60-min biodistribution data of 99mTc-3P-RGD2 using the same animal model with similar tumor size. Figure 4 compares the organ uptake and T/B ratios for 99mTcO(MAG2-3P-RGD2) and 99mTc-3P-RGD2 at 60 min p.i. Replacing [99mTc(HYNIC)(tricine)(TPPTS)] with the much smaller 99mTcO(MAG2) resulted in a significant increase in the tumor and normal organ uptake. For example, 99mTcO(MAG2-3P-RGD2) had the 60-min uptake of 15.48 ± 2.49, 1.40 ± 0.67, 9.39 ± 1.69, 13.38 ± 2.07, 3.87 ± 0.51 and 5.29 ± 0.79 %ID/g in the tumor, blood, intestine, kidneys, liver and lungs, respectively, while the uptake of 99mTc-3P-RGD2 in the same organs was 9.15 ± 2.13, 0.27 ± 0.08, 5.25 ± 1.92, 8.22 ± 1.99, 1.69 ± 0.46 and 2.10 ± 0.54 %ID/g at the same time point.

Figure 4.

Figure 4

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A: Biodistribution data of 99mTcO(MAG2-3P-RGD2) in athymic nude mice (n = 5) bearing U87MG human glioma xenografts in the absence/presence of excess E[c(RGDfK)]2 (14 mg/kg or ∼350 μg/mouse) at 60 min p.i. B: Direct comparison of biodistribution characteristics between 99mTcO(MAG2-3P-RGD2) and 99mTcO(MAG2-3P-RGK2) in athymic nude mice (n = 5) bearing U87MG human glioma xenografts at 60 min p.i.

Integrin αvβ3 Specificity

Figure 6A compares the organ uptake of 99mTcO(MAG2-3P-RGD2) in the absence/presence of E[c(RGDfK)]2 at 60 min p.i. Co-injection of excess E[c(RGDfK)]2 almost completely blocked its tumor uptake (0.31 ± 0.07 %ID/g with E[c(RGDfK)]2 vs. 15.48 ± 2.49 %ID/g without E[c(RGDfK)]2). The normal organ uptake of 99mTcO(MAG2-3P-RGD2) was also almost completely blocked by co-injection of excess E[c(RGDfK)]2.

Figure 6.

Figure 6

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Typical radio-HPLC chromatograms of 99mTcO(MAG2-3P-RGD2) in saline before injection, urine, feces, kidney, liver and tumor at 120 min p.i. Slight variation in HPLC retention time was probably caused by the presence of 20% acetonitrile in the organ extract.

RGD Specificity

Figure 6B compares the 60-min organ uptake values for 99mTcO(MAG2-3P-RGK2) and 99mTcO(MAG2-3P-RGD2). This experiment was designed to demonstrate the RGD-specificity of 99mTcO(MAG2-3P-RGD2). 99mTcO(MAG2-3P-RGK2) had much lower tumor uptake (0.70 ± 0.09 %ID/g) than 99mTcO(MAG2-3P-RGD2) (15.48 ± 2.49 %ID/g). It also had significantly (p < 0.01) lower uptake in normal organs than 99mTcO(MAG2-3P-RGD2). As a result, the radioactivity was excreted much faster (more urine activity at the same time point) from the renal route in the tumor-bearing mice administered with 99mTcO(MAG2-3P-RGK2).

Tumor Size vs. Tumor Uptake

In this study, we used a total of 14 glioma-bearing mice to explore the relationship between the tumor size and %ID uptake of 99mTcO(MAG2-3P-RGD2) at 120 min p.i. As illustrated in Figure 5A, there was a linear relationship between the tumor size (0.08 – 0.4 g; n = 14) and the %ID glioma uptake of 99mTcO(MAG2-3P-RGD2) with R2 = 0.9226. Obviously, the %ID tumor uptake of 99mTcO(MAG2-3P-RGD2) increased in a linear fashion as the tumor size became larger.

Figure 5.

Figure 5

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A: the relationship between tumor size and tumor uptake for 99mTcO(MAG2-3P-RGD2) at 120 min p.i. in the athymic nude mice (n = 14) bearing the U87MG glioma xenografts. The linear relationship between the tumor size and %ID tumor uptake suggests that 99mTcO(MAG2-3P-RGD2) has the potential for noninvasive monitoring of tumor growth or shrinkage during anti-angiogenic therapy. B: planar images of the athymic nude mice (bearing U87MG glioma xenografts) administered with ∼500 μCi of 99mTcO(MAG2-3P-RGD2) at 15, 30, 60 and 120 min p.i. Arrows indicate the presence of the tumor, gallbladder, kidneys and bladder.

Planar Imaging

Figure 7 illustrates representative static images of the glioma-bearing mice (n = 3) administered with 99mTcO(MAG2-3P-RGD2) at 15, 30, 60 and 120 min p.i. The tumor was clearly seen with excellent contrast as early as 15 min p.i. The tumor radioactivity remained relatively steady over the 2 h study period. The radioactivity accumulation in the blood and muscle disappeared almost completely by 120 min p.i. The most visible organs were tumor, gallbladder, kidneys and bladder. Because of the radioactivity accumulation in the abdominal region, it was difficult to accurately determine the tumor/kidney and tumor/liver ratios on the basis of planar imaging.

Metabolic Properties

Metabolism studies were performed on 99mTcO(MAG2-3P-RGD2) using athymic nude mice bearing U87MG glioma xenografts. The percentage of radioactivity recovery in the urine and feces samples was very high (>90%); but it was only 45 – 55% from the homogenates of the tumor, kidney and liver tissues. Figure 8 shows radio-HPLC chromatograms of 99mTcO(MAG2-3P-RGD2) in saline before injection, in the extracts of urine and feces samples, and in the homogenates from tumor, kidneys, and liver at 120 min p.i. There was no significant metabolism for 99mTcO(MAG2-3P-RGD2) during its excretion from renal and hepatobiliary routes, and in the tissues from tumor, liver and kidneys.

Discussion

In this study, we prepared two cyclic RGD dimer conjugates (MAG2-P-RGD2and MAG2-3P-RGD2). It is found that both 99mTcO(MAG2-P-RGD2) and 99mTcO(MAG2-3P-RGD2), can be obtained in high yield (>90%) and with high specific activity (∼5 Ci/μmol) using a kit formulation, and have very high solution stability in the kit matrix. There are advantages of using small peptides, such as MAG2 (the smallest N3S triamidethiol chelating unit when conjugated to the peptide) as BFCs. The attachment of BFC can be easily incorporated into solid-phase peptide synthesis. The N3S chelating units form stable Tc complexes with the [Tc=O]3+ core. The hydrophilicity of the Tc chelate can be tuned by changing the glycine residues with more hydrophilic amino acids, such as aspartic and glutamic acids.

The integrin αvβ3 binding affinity follows the order of MAG2-3P-RGD2 (IC50 = 3.9 ± 0.4 nM) > MAG2-P-RGD2 (IC50 = 8.6 ± 2.8 nM) > c(RGDfK) (IC50 = 46.6 ± 4.5 nM), suggesting that bivalency is most likely responsible for the higher integrin αvβ3 binding affinity of MAG2-3P-RGD2 as compared to that of MAG2-P-RGD2. This conclusion is well supported by the significantly (p < 0.01) higher tumor uptake (Figure 3) of 99mTcO(MAG2-3P-RGD2) than that of 99mTcO(MAG2-P-RGD2). If they were bound to integrin αvβ3 in the same fashion, MAG2-3P-RGD2 and MAG2-P-RGD2 would have had the same integrin αvβ3 binding affinity whereas 99mTcO(MAG2-3P-RGD2) and 99mTcO(MAG2-P-RGD2) would have shared the similar tumor uptake in the same tumor-bearing animal model.

Figure 3.

Figure 3

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Comparison of biodistribution data of 99mTcO(MAG2-3P-RGD2) and 99mTc-3P-RGD2 in the tumor, intestine, kidneys and liver from athymic nude mice (n = 5) bearing U87MG glioma xenografts at 60 min p.i.

The integrin αvβ3 binding affinity of MAG2-3P-RGD2 (IC50 = 3.9 ± 0.4 nM) is very similar to that of HYNIC-3P-RGD2 (IC50 = 4.1 ± 0.3 nM), respectively, suggesting that replacing HYNIC with MAG2 has little impact on the integrin αvβ3 binding affinity of 3P-RGD2. It must be noted that this comparison is between the cyclic RGD conjugates, not their 99mTc radiotracers, the integrin αvβ3 binding affinity and biodistribution characteristic of which could be altered significantly depending on the size of 99mTc chelates (Figure 1: 99mTcO(MAG2) vs. [99mTc(HYNIC)(tricine)(TPPTS)]). 99mTcO(MAG2) has a molecular weight of ∼300 Daltons, and is expected to have minimal impact on integrin αvβ3 binding affinity of 3PEG4-dimer. In contrast, [99mTc(HYNIC)(tricine)(TPPTS)] has a molecular weight of ∼1000 Daltons due to the presence of tricine and TPPTS coligands, and is expected to have much more significant impact on the integrin αvβ3 binding affinity of 3P-RGD2. This assumption seems to be supported by the significantly (p < 0.01) higher tumor uptake of 99mTcO(MAG2-3P-RGD2) (15.48 ± 2.49 %ID/g at 60 min p.i.) than that of 99mTc-3P-RGD2 (9.15 ± 2.13 at 60 min p.i.).

Replacing [99mTc(HYNIC)(tricine)(TPPTS)] with 99mTcO(MAG2) results in >10× higher lipophilicity for 99mTcO(MAG2-3P-RGD2) (log P = -3.19 ± 0.13) as compare to 99mTc-3P-RGD2 (log P = -4.35 ± 0.10), which is most likely responsible for its higher uptake in tumor, blood and normal organs (Figure 4: top). For example, the blood radioactivity for 99mTcO(MAG2-3P-RGD2) is 1.40 ± 0.67 %ID/g at 60 min p.i. while it is only 0.27 ± 0.08 %ID/g for 99mTc-3P-RGD2 at the same time point. The liver uptake of 99mTcO(MAG2-3P-RGD2) is 3.87 ± 0.51 %ID/g whereas 99mTc-3P-RGD2 has the liver uptake of 2.92 ± 0.77 %ID/g at 60 min p.i. 99mTcO(MAG2-3P-RGD2) has the kidney uptake of 13.38 ± 2.07 %ID/g while 99Tc-3PEG4-dimer has the kidney uptake of 8.62 ± 1.99 %ID/g at 60 min p.i. The lung uptake is 5.29 ± 0.79 %ID/g for 99mTcO(MAG2-3P-RGD2) and 2.10 ± 0.54 %ID/g for 99mTc-3P-RGD2 at 60 min p.i. As a result, the tumor/blood, tumor/liver, tumor/lung and tumor/liver ratios for 99mTcO(MAG2-3P-RGD2) are not as good as those for 99mTc-3P-RGD2 (Figure 4: bottom). On the basis of these data, we believe that 99mTc-3P-RGD2 remains to be a better SPECT radiotracer for noninvasive imaging of integrin αvβ3–positive tumors.

The integrin αvβ3-specificity of 99mTcO(MAG2-3P-RGD2) has been demonstrated by the “blocking experiment” (Figure 6A). The blockage of its tumor uptake indicates that 99mTcO(MAG2-3P-RGD2) is integrin αvβ3-specific. The uptake blockage in the eyes, heart, intestine, lungs, liver and spleen suggests that its accumulation in these organs is also integrin αvβ3-mediated. This conclusion is supported by the immunohistopathological studies (34, 35), which showed a strong positive staining of endothelial cells of small glomeruli vessels in the kidneys and weak staining in branches of the hepatic portal vein. The RGD-specificity of 99mTcO(MAG2-3P-RGD2) is demonstrated by the lower integrin αvβ3 binding affinity of MAG2-3P-RGK2 (IC50 = 711 ± 128 nM) than that of MAG2-3P-RGD2 (IC50 = 3.9 ± 0.4 nM), and the lower tumor uptake of 99mTcO(MAG2-3P-RGK2) than that of 99mTcO(MAG2-3P-RGD2) (Figure 6B). In addition, 99mTcO(MAG2-3PEG4-dimer) is able to maintain its chemical integrity during excretion from both renal and hepatobiliary routes, and in the tumor and liver tissues (Figure 8).

The ability to non-invasively quantify the integrin αvβ3 level provides new opportunities to select appropriate patients for anti-angiogenic treatment of integrin αvβ3-positive cancer patients (59, 60). The %ID tumor uptake reflects the total integrin αvβ3 level while the %ID/g tumor uptake reflects the integrin αvβ3 density. When tumor is small (<0.05 g or 50 m3), there is little angiogenesis with low blood flow. As a result, 99mTcO(MAG2-3P-RGD2) has low %ID tumor uptake (Figure 5A). As tumor grows, the integrin αvβ3 level becomes higher, and the %ID tumor uptake of 99mTcO(MAG2-3P-RGD2) increases (Figure 5A). The linear relationship between the tumor size and %ID tumor uptake strongly suggests that 99mTcO(MAG2-3P-RGD2) has the potential for monitoring the integrin αvβ3 expression levels during anti-angiogenic therapy. It is important to note that the integrin αvβ3 expression is not homogenous in the tumor tissue. Parts of the tumor tissue may become necrotic when its size is >10 mm in diameter. Therefore, the radiotracer %ID/g uptake in the tumors of different size is scattered as previously reported for 99mTc-3P-RGD2 (55), 99mTc-3G-RGD2 (56), and 64Cu(DOTA-3P-RGD2) (57).

We must be concerned that the subcutaneous glioma-bearing model used in this study may not reflect the real tumor growth rate in glioma cancer patients since the tumor growth is too fast in the athymic nude mice bearing U87MG glioma xenografts. However, this tumor-bearing animal model as a screening tool should provide us sufficient biodistribution data to select an optimal radiotracer for more preclinical evaluations in the future. In addition, we also noticed that the tumor growth rate is largely dependent on the number of U87MG human glioma cells implanted in each animal, and the radiotracer tumor uptake may vary significantly with the tumor growth rate. Whenever possible, the comparison between different radiotracers should be made by using their biodistribution data obtained from the same groups of tumor-bearing mice with similar tumor size. That is why in this study we obtained the 60-min biodistribution data of 99mTc-3PEG4-dimer, instead of using those reported in our previous studies (55), to demonstrate the impact of 99mTc chelates (99mTcO(MAG2) vs. [99mTc(HYNIC)(tricine)(TPPTS)]) on radiotracer tumor uptake and T/B ratios.

Conclusion

In this study, we presented the synthesis of two cyclic RGD peptide conjugates, MAG2-P-RGD2 and MAG2-3P-RGD2, and evaluated 99mTcO(MAG2-P-RGD2) and 99mTcO(MAG2-3P-RGD2) as new radiotracers for noninvasive imaging of integrin αvβ3 expression in athymic nude mice bearing U87MG glioma xenografts. The key findings are: (1) MAG2 is such an efficient BFC that 99mTcO(MAG2-3P-RGD2) could be readily prepared in high yield with high specific activity using a single-vial kit formulation; (2) replacing [99mTc(HYNIC)(tricine)(TPPTS)] with 99mTcO(MAG2) results in a significant increase in the radiotracer uptake in the tumor, blood and normal organs due to the increased lipophilicity of 99mTcO(MAG2-3P-RGD2). Even though 99mTcO(MAG2-3P-RGD2) has the better tumor uptake, its tumor/blood, tumor/liver, tumor/lung and tumor/liver ratios are not as good as those for 99mTc-3P-RGD2. On the basis of these results, we believe that future research should be directed towards BFC with highly charged amino acid residues, such as aspartic and glutamic acids, instead of glycine in MAG2.

Acknowledgments

This work is supported, in part, by Purdue University and research grants: R01 CA115883 A2 (S.L.) from National Cancer Institute (NCI), R21 HL083961-01 (S.L.) from the National Heart, Lung, and Blood Institute (NHLBI), and DE-FG02-08ER64684 (S.L.) from the Department of Energy, and NSF-30780728 (F.W.) from the National Science Foundation of China.

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