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. Author manuscript; available in PMC: 2011 May 19.
Published in final edited form as: Bioconjug Chem. 2010 May 19;21(5):969–978. doi: 10.1021/bc900555q

Evaluation of 111In-Labeled Cyclic RGD Peptides: Tetrameric Not Tetravalent

Sudipta Chakraborty 1, Jiyun Shi 1,2, Young-Seung Kim 1, Yang Zhou 1, Bing Jia 2, Fan Wang 2, Shuang Liu 1,*
PMCID: PMC2874107  NIHMSID: NIHMS196211  PMID: 20387808

Abstract

This report presents the synthesis and evaluation of 111In(DOTA-6G-RGD4) (DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid; 6G-RGD4 = E{G3-E[G3-c(RGDfK)]2}2 and G3 = Gly-Gly-Gly), 111In(DOTA-RGD4) (RGD4 = E{E[c(RGDfK)]2}2) and 111In(DOTA-3G-RGD2) (3G-RGD2 = G3-E[G3-c(RGDfK)]2) as new radiotracers for imaging integrin αvβ3–positive tumors. The IC50 values of DOTA-6G-RGD4, DOTA-RGD4 and DOTA-3G-RGD2 were determined to be 0.4 ± 0.1, 1.5 ± 0.2 and 1.3 ± 0.2 nM against 125I-c(RGDyK) bound to integrin αvβ3–positive U87MG human glioma cells. 111In(DOTA-6G-RGD4), 111In(DOTA-RGD4) and 111In(DOTA-3G-RGD2) were prepared by reacting 111InCl3 with the respective DOTA conjugate in NH4OAc buffer (100 mM, pH = 5.5). Radiolabeling could be completed by heating the reaction mixture at 100 °C for 15 – 20 min. The specific activity was ~1850 MBq/μmol for 111In(DOTA-3G-RGD2) and ~1480 MBq/μmol for 111In(DOTA-6G-RGD4). The athymic nude mice bearing U87MG human glioma xenografts were used to evaluate tumor uptake and excretion kinetics of 111In(DOTA-6G-RGD4), 111In(DOTA-RGD4) and 111In(DOTA-3G-RGD2). The results from both the integrin αvβ3 binding assay and biodistribution studies suggest that the tetrameric cyclic RGD peptides, such as RGD4 and 6G-RGD4, are most likely bivalent in binding to the integrin αvβ3. Both 111In(DOTA-6G-RGD4) and 111In(DOTA-RGD4) had significantly higher tumor uptake than 111In(DOTA-3G-RGD2) at 24 – 72 h post-injection due to the extra RGD motifs in RGD4 and 6G-RGD4. 111In(DOTA-3G-RGD2) had very little metabolism while 111In(DOTA-6G-RGD4) had a significant metabolism during its excretion via both renal and hepatobiliary routes over the 2 h period, probably due to its much larger size. The combination of high tumor uptake with long tumor retention suggests that their corresponding 90Y and 177Lu analogs M(DOTA-6G-RGD4) (M = 90Y and 177Lu) might be useful as therapeutic radiotracers for treatment of integrin αvβ3-positive solid tumors.

Keywords: integrin αvβ3, 111In-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 activated endothelial cells of the neovasculature during tumor growth, invasion and metastasis (1–9). Over last several years, many multimeric cyclic RGD peptides, such as E[c(RGDfK)]2 (RGD2) and E{E[c(RGDfK)]2}2 (RGD4), have been used to maximize the integrin αvβ3 binding affinity and improve the radiotracer tumor uptake (10–30). The results from in vitro assays, ex-vivo biodistribution and in vivo imaging studies clearly demonstrated that radiolabeled (99mTc, 18F, 64Cu and 68Ga) cyclic RGD peptide tetramers, such as E{E[c(RGDxK)]2}2 (x = f and y), have better tumor targeting capability as evidenced by their higher tumor uptake and longer tumor retention times than their dimeric and monomeric counterparts (25, 26, 29, 30). In our previous studies (31–37), we found that RGD2 was monodentate while PEG4-E[PEG4-c(RGDfK)]2 (3P-RGD2: PEG4 = 15-amino-4,7,10,13-tetraoxapentadecanoic acid) and (G3-E[G3-c(RGDfK)]2 (3G-RGD2: G3 = Gly-Gly-Gly) and are bivalent due to the longer distance between two RGD motifs. However, it remains unclear if the cyclic RGD tetramers, such as RGD4, are tetravalent in binding to integrin αvβ3.

To answer this fundamental question, we prepared a new RGD tetramer conjugate, DOTA-E{G3-E[G3-c(RGDfK)]2}2 (Figure 1: DOTA-6G-RGD4: DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) and its 111In complex 11In(DOTA-6G-RGD4). A competitive displacement assay was used to determine the integrin αvβ3 binding affinity of DOTA-3G-RGD2, DOTA-RGD4 and DOTA-6G-RGD4 against 125I-c(RGDyK) bound to the integrin αvβ3–positive U87MG glioma cells. Biodistribution and imaging studies were carried out in athymic nude mice bearing U87MG glioma xenografts. For comparison purposes, we also evaluated DOTA-RGD4, DOTA-3G-RGD2,111In(DOTA-RGD4) and 111In(DOTA-3G-RGD2) using the same assays. The comparison of IC50 values of DOTA-3G-RGD2, DOTA-RGD4 and DOTA-6G3-RGD4 and tumor uptake of 111In(DOTA-6G-RGD4), 111In(DOTA-RGD4) and 111In(DOTA-3G-RGD2) will allow us to determine the valency of RGD4 and 6G-RGD4.

Figure 1.

Figure 1

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Structure of a new cyclic RGD tetramer conjugate: DOTA-6G-RGD4.

EXPERIMENTAL SECTION

Materials and Instruments

Chemicals were purchased from Sigma/Aldrich (St. Louis, MO). Cyclic RGD peptide 3G-RGD2 was purchased from the Peptides International, Inc. (Louisville, KY). DOTA-OSu (1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid-1-acetate(N-hydroxysuccinamide)) was purchased from Macrocyclics (Dallas, TX). DOTA-3G-RGD2 and DOTA-RGD4 were prepared according to the literature methods (14, 26, 33). MALDI (Matrix Assisted Laser Desorption Ionizations) Mass spectral data were collected using an Applied Biosystems Voyager DE PRO mass spectrometer (Framingham, MA), Department of Chemistry, Purdue University. 111InCl3 was obtained from Perkin-Elmer Life and Analytical Sciences (North Billerica, MA). The NH4OAc buffer for 111In-labeling studies was passed over a Chelex-100 column (1×15 cm) to minimize the trace metal contaminants.

HPLC Methods

Method 1 used a LabAlliance HPLC system equipped with a UV/vis detector (λ=254 nm) and Zorbax C18 semi-prep column (9.4 mm × 250 mm, 100 Å pore size; Agilent Technologies, Santa Clara, CA). The flow rate was 2.5 mL/min and the mobile phase was isocratic with 90% A (0.1% TFA in water) and 10% B (0.1% TFA in acetonitrile) at 0–5 min, followed by a gradient mobile phase going from 10% B at 5 min to 40% B at 20 min. 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 – 5 min, followed by a gradient mobile phase from 10% B at 5 min to 20% B at 15 min and to 50% B at 20 min.

Boc-E{G3-E[G3-c(RGDfK)]2}2 (Boc-6G-RGD4)

The succinimidyl ester of Boc-protected glutamic acid (Boc-E(OSu)2) was prepared according to the literature method (12). To a solution of Boc-E(OSu)2 (2.1 mg, 4.75 μmol) in DMF (2 mL) was added 3G-RGD2 (21 mg, 11.5 μmol). After addition of DIEA (3 drops), the reaction mixture was stirred for 2 h at room temperature. The reaction was terminated by addition of 3 mL NH4OAc buffer (100 mM, pH = 7.0). The product was isolated by HPLC (Method 1). Lyophilization of the collected fractions at 15.8 min afforded desired product Boc-6G-RGD4. The yield was 7.6 mg (~41%). MALDI-MS: m/z = 3877 for [M + H]+ (M = 3875 calcd. for [C164H241N57O54]+).

E{G3-E[G3-c(RGDfK)]2}2 (6G-RGD4)

Boc-6G-RGD4 was dissolved in TFA (2 mL). After standing at room temperature for 5 min, excess TFA was removed. The residue was dissolved in 2 mL of 0.1 M NH4OAc buffer (100 mM, pH = 7.0). The product was separated from the mixture by HPLC (Method 1). Lyophilization of the collected fractions at 14.2 min afforded 6G-RGD4. The yield was 5.6 mg (~76%). MALDI-MS: m/z = 3777 for [M + H]+ (M = 3775 calcd. for [C159H233N57O52]+).

DOTA-E{G3-E[G3-c(RGDfK)]2}2 (DOTA-6G-RGD4)

DOTA-OSu (5.0 mg, 10.0 μmol) and 6G-RGD4 (3.8 mg, 1.0 μmol) were dissolved in anhydrous DMF (1 mL). After addition of excess DIEA (3 drops), the reaction mixture was stirred for 2 h at room temperature. Upon addition of 2 mL NH4OAc buffer (100 mM, pH = 7.0), the product was separated by HPLC (Method 1). Lyophilization of the collected fractions at 14.5 min afforded DOTA-6G-RGD4 with >95% purity. The yield was 3.2 mg (~76%). MALDI-MS: m/z = 4163 for [M + H]+ (M = 4161 calcd. for [C175H259N61O59]+).

111In-Labeling and Dose Preparation

To a clean Eppendorf tube were added 200–300 μL of the DOTA conjugate solution (0.1 mg/mL in 0.1 M NaOAc buffer, pH = 5.5) and 20 μL of 111InCl3 solution (~18.5 MBq in 0.05 M HCl). The vial was heated in a water bath at 100 °C for ~ 15 min. After heating, the Eppendorf tube was allowed to stand at room temperature for ~10 min. A sample of the resulting solution was analyzed by radio-HPLC (Method 2). For biodistribution studies, the 111In radiotracer was purified by HPLC (Method 2). Volatiles in the mobile phase were removed under vacuum (~5 mmHg) at room temperature. Doses were prepared by dissolving the purified radiotracer in saline to a concentration of ~1.1 MBq/mL. For imaging studies, doses were prepared by dissolving the radiotracer in saline to ~37 MBq/mL. The resulting solution was filtered with a 0.20 micron Millex-LG filter before being injected into animals. Each animal was injected with ~0.1 mL of the dose solution.

Determination of Octanol/Water Partition Coefficients

The octanol/water partition coefficients were determined using the following procedure. In short, the 111In 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 both n-octanol and aqueous layers were counted in a Perkin Elmer Wizard – 1480 γ-counter (Shelton, CT). The partition coefficients were calculated. The log P values were 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 and specificity of RGD peptides were assessed using 125I-c(RGDyK) as the integrin-specific radioligand. Briefly, U87MG human 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 glioma cells in binding buffer and incubated with 125I-c(RGDyK) in the presence of increasing concentrations of the RGD conjugate. After removing the unbound 125I-c(RGDyK), hydrophilic PVDF filters were collected. The radioactivity was determined using a γ-counter (Packard, Meriden, CT). The IC50 values were calculated by fitting the data by nonlinear regression using GraphPad Prism™ (GraphPad Software, Inc., San Diego, CA). Experiments were carried out twice with triplicate samples. The IC50 values are reported as an average plus the standard deviation.

Animal Model

Biodistribution and imaging studies were performed using the athymic nude mice bearing U87MG human glioma xenografts in compliance NIH animal experiment guidelines (Principles of Laboratory Animal Care, NIH Publication No. 86-23, revised 1985). The animal protocol was approved by the Purdue University Animal Care and Use Committee. U87MG cells were cultured in the Minimum Essential Medium, Eagle with Earle’s Balanced Salt Solution (non-essential amino acids sodium pyruvate) (ATCC, Manassas, VA) in humidified atmosphere of 5% carbon dioxide, and were supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin solution (GIBCO, Grand Island, NY). Female athymic nu/nu mice were purchased from Harlan (Indianapolis, IN) at 4 – 5 weeks of age. Each mouse was implanted subcutaneously with 5 × 106 tumor cells into the left and right upper flanks. In this way, one could assess the impact of tumor size on the radiotracer imaging quality in a single tumor-bearing mouse. All procedures will be performed in a laminar flow cabinet using aseptic technique. Three to four weeks after inoculation, the tumor size was 0.05 – 0.4 g, and animals were used for biodistribution and imaging studies.

Biodistribution Protocol

Twenty-five tumor-bearing mice (20 – 25 g) were randomly divided into five groups. Each tumor-bearing mouse was administered with ~111 KBq of the 111In radiotracer by tail vein injection. Five animals were sacrificed by sodium pentobarbital overdose (~200 mg/kg) at 0.5, 1, 4, 24 and 72 h postinjection (p.i.). Blood samples were withdrawn from the heart of the tumor-bearing mice. Tumor 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 γ-counter (Perkin Elmer Wizard – 1480, Shelton, CT). The organ uptake was calculated as the percentage of injected dose per gram of organ mass (%ID/g) or the percentage of injected dose per organ (%ID/organ). For the blocking experiment, five tumor-bearing athymic nude mice (20 – 25 g) were used, and each animal was administered with ~111 KBq of 111In(DOTA-6G-RGD4) along with ~350 μg (~14 mg/kg) of RGD2. Such a high dose of blocking agent was used to make sure that the integrin αvβ3 binding was completely blocked. At 1 h p.i., all five tumor-bearing animals were sacrificed for organ biodistribution. Biodistribution data (%ID/g) and target-to-background (T/B) ratios are reported as an average plus the standard variation based on the results from five tumor-bearing mice (10 tumors) at each time point. 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.

Planar Imaging

The athymic nude mice (n = 3) bearing U87MG glioma xenografts were used for imaging studies of 111In(DOTA-6G-RGD4) and 111In(DOTA-3G-RGD2). Each tumor-bearing mouse was administered with ~3.7 MBq of 111In radiotracer via tail vein injection. Animals were anesthetized with intraperitoneal injection of ketamine (80 mg/kg) and xylazine (19 mg/kg), and were then placed supine on a single head mini γ-camera (Diagnostic Services Inc., NJ) equipped with a parallel-hole, medium-energy and high-resolution collimator. Anterior images were acquired at 0.5, 1, 4, 24 and 72 h p.i. and stored digitally in a 128 × 128 matrix. The acquisition count limits were set at 200 K. After completion of imaging, animals were sacrificed by sodium pentobarbital overdose (~200 mg/kg).

Metabolism

Normal nude mice were used for metabolism studies. Each mouse was administered with ~3.7 MBq of 111In(DOTA-3G-RGD2) and 111In(DOTA-6G-RGD4) via 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 passed through a 0.20 μ Millex-LG filter unit. The filtrate was analyzed by HPLC (Method 2). Feces, liver and kidney samples were collected at 120 min p.i. and suspended in 20% acetonitrile aqueous solution after homogenization. The resulting mixture was vortexed for 5 – 10 min. After centrifuging at 8,000 rpm, the supernatant was collected and passed through a 0.20 μ Millex-LG filter unit. The filtrate was analyzed by radio-HPLC (Method 2). The percentage radioactivity recovery was >95% (by γ-counting) for both urine and feces samples.

RESULTS

DOTA-6G-RGD4

Synthesis of DOTA-6G-RGD4 was straightforward. The key step was the deprotection of Boc-6G-RGD4. The excess TFA must be removed within 10 min of the reaction. Otherwise, the major product was the hydrolyzed product E-3G-RGD2 (E-G3-E[G3-c(RGDfK)]2) not 6G-RGD4. Conjugation of 6G-RGD4 with excess DOTA-OSu in DMF in the presence of excess DIEA afforded DOTA-6G-RGD4 in high yield (76%). Its identity has been confirmed by MALDI-MS.

In Vitro Integrin αvβ3 Binding Affinity

The integrin αvβ3 binding affinities of c(RGDyK), DOTA-3G-RGD2, DOTA-RGD4 and DOTA-6G-RGD4 were determined using the integrin αvβ3–positive U87MG human glioma cells. Figure 2 shows displacement curves of 125I-c(RGDyK) by c(RGDyK), DOTA-3G-RGD2, DOTA-RGD4 and DOTA-6G-RGD4. Their IC50 values were calculated to be 42.2 ± 5.2, 1.3 ± 0.2, 1.5 ± 0.2 and 0.4 ± 0.1 nM, respectively.

Figure 2.

Figure 2

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Competitive inhibition curves of 125I-c(RGDyK) bound to the U87MG human glioma cells by c(RGDyK), DOTA-3G-RGD2, DOTA-RGD4 and DOTA-6G3RGD4. Their IC50 values were calculated to be 42.2 ± 5.2, 1.1 ± 0.1, 1.4 ± 0.3 and 0.4 ± 0.1 nM, respectively.

Radiochemistry

111In(DOTA-6G-RGD4), 111In(DOTA-RGD4) and 111In(DOTA-3G-RGD2) were prepared by reacting 111InCl3 with DOTA-6G-RGD4, DOTA-RGD4 and DOTA-3G-RGD2, respectively, in NH4OAc buffer (100 mM, pH = 5.5). Radiolabeling was completed by heating the reaction mixture at 100 °C for ~15 min. The radiochemical purity was >92% for all three 111In radiotracers. The specific activity was ~925 MBq/mg or (~1850 MBq/μmol) for 111In(DOTA-3G-RGD2). They were analyzed using the same method (Method 2). Their HPLC retention times and log P values were listed in Table 1.

Table 1.

Radiochemical purity (RCP), HPLC retention time and log P values for 111In-labeled cyclic RGD peptides.

Compound RCP (%) Retention Time (min) Log P Value
111In(DOTA-3G-RGD2) >95 13.9 −4.13 ± 0.08
111In(DOTA-6G-RGD4) > 92 17.1 −4.40 ± 0.04
111In(DOTA-RGD4) > 92 19.1 −3.41 ± 0.13
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Biodistribution Characteristics

Selected biodistribution data are listed in Tables 2–4 for 111In(DOTA-6G-RGD4), 111In(DOTA-RGD4) and 111In(DOTA-3G-RGD2). Figure 3 compares their uptake (%ID/g) in the tumor and intestine, as well as their tumor/kidney and tumor/liver ratios in the athymic nude mice bearing U87MG human glioma xenografts. The comparison of their tumor uptake in combination with the IC50 values of cyclic RGD peptide DOTA conjugates will allow us to determine if they are actually tetravalent in binding to the integrin αvβ3.

Table 2.

Selected biodistribution data of 111In(DOTA-6G-RGD4) in athymic nude mice (n = 5) bearing U87MG human glioma xenografts.

%ID/gram 0.5 h 1 h 4 h 24 h 72 h
Blood 0.97 ± 0.10 0.25 ± 0.04 0.05 ± 0.00 0.06 ± 0.02 0.03 ± 0.01
Bone 2.26 ± 0.71 2.35 ± 0.64 1.99 ± 0.15 1.41 ± 0.29 0.90 ± 0.41
Brain 0.13 ± 0.08 0.14 ± 0.03 0.13 ± 0.07 0.12 ± 0.07 0.10 ± 0.04
Eyes 1.20 ± 0.60 1.54 ± 0.22 1.61 ± 1.38 0.83 ± 0.20 0.53 ± 0.12
Heart 1.50 ± 0.76 1.49 ± 0.29 1.03 ± 0.23 0.80 ± 0.22 0.51 ± 0.16
Intestine 7.15 ± 3.70 9.25 ± 1.27 8.79 ± 2.98 10.91 ± 1.94 8.31 ± 2.14
Kidney 8.35 ± 0.52 8.42 ± 0.86 6.21 ± 0.65 4.44 ± 0.45 3.45 ± 0.90
Liver 3.04 ± 1.38 3.77 ± 0.79 3.05 ± 0.14 2.46 ± 0.36 2.01 ± 0.45
Lungs 3.65 ± 0.87 3.44 ± 1.86 2.72 ± 0.38 1.51 ± 0.22 0.91 ± 0.08
Muscle 1.23 ± 0.42 1.14 ± 0.23 0.80 ± 0.11 0.93 ± 0.54 0.45 ± 0.06
Spleen 2.63 ± 0.45 2.38 ± 0.49 2.35 ± 0.23 1.96 ± 0.31 2.07 ± 0.24
U87MG 12.66 ± 1.25 11.45 ± 2.46 8.99 ± 2.61 8.68 ± 1.42 5.59 ± 0.54
Tumor/Blood 13.06 ± 1.29 44.92 ± 9.66 198.34 ± 57.67 143.01 ± 23.40 152.37 ± 102.84
Tumor/Bone 5.60 ± 0.55 4.88 ± 1.05 4.53 ± 1.32 6.16 ± 1.01 4.67 ± 3.15
Tumor/Kidney 1.52 ± 0.15 1.36 ± 0.29 1.45 ± 0.42 1.95 ± 0.32 1.21± 0.82
Tumor/Liver 4.16 ± 0.41 3.04 ± 0.65 2.95 ± 0.86 3.53 ± 0.58 2.09 ± 1.41
Tumor/Lungs 3.46 ± 0.34 3.33 ± 0.72 3.30 ± 0.96 5.74 ± 0.94 4.59 ± 3.10
Tumor/Muscle 10.30 ± 1.02 10.08 ± 2.17 11.20 ± 3.26 9.30 ± 1.52 9.21± 6.22
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Table 4.

Biodistribution data of 111In(DOTA-3G-RGD2) in athymic nude mice (n = 5) bearing U87MG human glioma xenografts.

%ID/gram 0.5 h 1 h 4 h 24 h 72 h
Blood 0.87 ± 0.13 0.30 ± 0.07 0.09 ± 0.00 0.03 ± 0.01 0.01 ± 0.00
Brain 0.13 ± 0.03 0.09 ± 0.00 0.07 ± 0.00 0.05 ± 0.01 0.04 ± 0.00
Eyes 1.20 ± 0.24 0.84 ± 0.13 0.51 ± 0.04 0.32 ± 0.05 0.16 ± 0.03
Heart 1.03 ± 0.12 0.60 ± 0.12 0.44 ± 0.02 0.28 ± 0.04 0.15 ± 0.04
Intestine 4.83 ± 1.15 4.81 ± 1.01 5.09 ± 1.75 3.77 ± 0.07 1.89 ± 0.19
Kidney 6.74 ± 0.79 3.88 ± 0.48 2.69 ± 0.10 2.08 ± 0.16 1.48 ± 0.14
Liver 2.45 ± 0.14 2.25 ± 0.04 2.23 ± 0.05 1.52 ± 0.15 0.85 ± 0.09
Lungs 3.13 ± 0.47 1.71 ± 0.48 1.22 ± 0.08 0.75 ± 0.02 0.32 ± 0.06
Muscle 1.33 ± 0.24 0.70 ± 0.03 0.39 ± 0.05 0.30 ± 0.09 0.20 ± 0.05
Spleen 1.63 ± 0.08 1.36 ± 0.13 1.18 ± 0.20 0.91 ± 0.10 0.88 ± 0.17
U87MG 10.19 ± 3.61 10.69 ± 1.82 6.72 ± 2.06 4.83 ± 1.19 1.99 ± 0.39
Tumor/Blood 11.74 ± 3.82 36.93 ± 9.74 72.50 ± 23.09 300.15 ± 29.08 215.91 ± 56.80
Tumor/Kidney 1.51 ± 0.47 2.77 ± 0.51 2.49 ± 0.75 2.36 ± 0.69 1.86 ± 0.30
Tumor/Liver 4.16 ± 1.43 4.74 ± 0.81 3.01 ± 0.87 3.25 ± 1.01 2.35 ± 0.54
Tumor/Lungs 3.35 ± 1.36 6.59 ± 1.86 5.60 ± 1.93 6.38 ± 1.49 6.43 ± 1.66
Tumor/Muscle 8.03 ± 3.56 15.31 ± 2.44 17.61 ± 5.95 17.86 ± 7.44 10.79 ± 3.69
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Figure 3.

Figure 3

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Direct comparison of the tumor and intestine uptake (%ID/g), tumor/kidney and tumor/liver ratios between 111In(DOTA-6G-RGD4), 111In(DOTA-RGD4) and 111In(DOTA-3G-RGD2) in the athymic nude mice (n = 5) bearing U87MG human glioma xenografts.

In general, 111In(DOTA-6G-RGD4) excreted predominantly via the renal route with >75% of the injected radioactivity recovered from urine and <10% of the injected radioactivity recovered from feces at 2 h p.i. 111In(DOTA-6G-RGD4) had a rapid blood clearance with high tumor/blood ratios (Table 2). It had a high initial tumor uptake (12.66 ± 1.25 %ID/g at 0.5 h p.i.) with a slow washout (11.45 ± 2.46, 8.99 ± 2.61, 6.68 ± 1.42 and 5.59 ± 0.54 %ID/g at 1, 4, 24 and 72 h p.i., respectively). The liver uptake of 111In(DOTA-6G-RGD4) was low (3.04 ± 1.38, 3.77 ± 0.79, 3.05 ± 0.14, 2.46 ± 0.36 and 2.01 ± 0.45 %ID/g at 0.5, 1, 4, 24 and 72 h p.i., respectively), and its tumor/liver ratios were high (4.16 ± 0.41, 3.04 ± 0.65, 2.95 ± 0.86, 3.53 ± 0.58 and 2.09 ± 1.41 at 0.5, 1, 4, 24 and 72 h p.i., respectively). The kidney and muscle uptake of 111In(DOTA-6G-RGD4) was also low with high tumor/kidney and tumor/muscle ratios (Table 2). The tumor uptake of 111In(DOTA-RGD4) was high (8.97 ± 0.52, 7.98 ± 2.36, 7.32 ± 2.66, 5.84 ± 1.09 and 3.16 ± 0.43 %ID/g at 0.5, 1, 4, 24 and 72 h p.i., respectively), but its tumor retention time was not as long as that of 111In(DOTA-6G-RGD4) (Figure 3). 111In(DOTA-RGD4) had a rapid blood clearance with high tumor/blood ratios over the 72 h period (Table 3). The initial kidney uptake (Figure 3) of 111In(DOTA-RGD4) was significantly higher than that of 111In(DOTA-6G-RGD4). However, there was no significant difference between 111In(DOTA-RGD4) and 111In(DOTA-6G-RGD4) in their kidney uptake at >1 h p.i. 111In(DOTA-RGD4) and 111In(DOTA-6G-RGD4) shared very similar liver uptake and tumor/liver ratio (Figure 3). 111In(DOTA-3G-RGD2) also had a rapid blood clearance with high tumor/blood ratios (Table 4). Its initial tumor uptake (10.19 ± 3.61 %ID/g at 0.5 h p.i.) was comparable to that of 111In(DOTA-RGD4) and 111In(DOTA-6G-RGD4) within the experimental error; but its tumor uptake was significantly (p < 0.01) lower (Figure 3: tumor uptake = 5.59 ± 0.54 %ID/g for 111In(DOTA-6G-RGD4), 3.16 ± 0.43 %ID/g for 111In(DOTA-RGD4) and 1.99 ± 0.39 %ID/g for 111In(DOTA-3G-RGD2) at 72 h p.i.). The uptake of 111In(DOTA-3G-RGD2) in intestine, kidneys and liver was also significantly (p < 0.05) lower than that of 111In(DOTA-RGD4) and 111In(DOTA-6G-RGD4). As a result, the tumor/kidney and tumor/liver ratios of 111In(DOTA-3G-RGD2) were higher than that of both 111In(DOTA-RGD4)and 111In(DOTA-6G-RGD4) (Figure 3).

Table 3.

Selected biodistribution data of 111In(DOTA-RGD4) in athymic nude mice (n = 5) bearing U87MG human glioma xenografts.

%ID/gram 0.5 h 1 h 4 h 24 h 72 h
Blood 0.80 ± 0.18 0.37 ± 0.08 0.12 ± 0.05 0.04 ± 0.01 0.07 ± 0.02
Bone 1.64 ± 0.17 0.93 ± 0.13 0.86 ± 0.20 0.53 ± 0.27 0.75 ± 0.09
Brain 0.15 ± 0.04 0.12 ± 0.01 0.10 ± 0.02 0.07 ± 0.02 0.14 ± 0.07
Eyes 1.56 ± 0.15 1.23 ± 0.10 0.92 ± 0.46 0.64 ± 0.10 0.78 ± 0.46
Heart 1.45 ± 0.53 1.23 ± 0.11 0.90 ± 0.18 0.54 ± 0.26 1.01 ± 0.76
Intestine 10.79 ± 1.46 8.42 ± 1.65 9.19 ± 1.58 7.03 ± 2.06 3.37 ± 0.30
Kidney 11.18 ± 1.22 7.28 ± 0.57 5.83 ± 0.70 5.11 ± 0.52 3.48 ± 1.10
Liver 2.98 ± 0.51 2.67 ± 0.32 2.75 ± 0.29 2.15 ± 0.57 1.62 ± 0.40
Lungs 4.29 ± 1.26 3.43 ± 0.54 2.06 ± 0.39 1.50 ± 0.39 0.62 ± 0.36
Muscle 1.79 ± 0.38 1.22 ± 0.45 0.63 ± 0.11 0.78 ± 0.19 0.68 ± 0.31
Spleen 2.65 ± 1.36 2.40 ± 0.51 1.40 ± 0.95 1.15 ± 0.84 1.16 ± 0.63
U87MG 8.97 ± 0.52 7.98 ± 2.36 7.32 ± 2.66 5.84 ± 1.09 3.16 ± 0.43
Tumor/Blood 10.61 ± 1.97 19.00 ± 3.60 42.74 ± 5.65 151.67 ± 57.32 31.78 ± 4.57
Tumor/Bone 5.35 ± 0.60 8.58 ± 1.46 9.22 ± 4.79 28.78 ± 16.18 4.49 ± 0.04
Tumor/Kidney 0.84 ± 0.05 1.09 ± 0.25 1.26 ± 0.36 1.15 ± 0.74 0.96 ± 0.22
Tumor/Liver 2.69 ± 0.29 2.85 ± 0.67 2.50 ± 0.60 2.91 ± 1.17 1.68 ± 0.23
Tumor/Lungs 2.32 ± 1.03 2.77 ± 1.14 4.06 ± 0.83 4.71 ± 1.79 5.45 ± 1.61
Tumor/Muscle 5.37 ± 0.20 6.50 ± 1.22 14.07 ± 5.93 9.31 ± 6.36 4.87 ± 2.24
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Tumor Size vs. Tumor Uptake

As illustrated in Figure 4A, there was a linear relationship between the %ID tumor uptake of 111In(DOTA-3G-RGD2) at 60 min p.i. and tumor size (0.03 – 0.60 g; n = 12) with R2 = 0.959. As the tumor became larger, the total tumor uptake of 111In(DOTA-3G-RGD2) increased. In contrast, the %ID/g tumor uptake (Figure 4B) of 111In(DOTA-3G-RGD2) decreased as the tumor size increased. For example, 111In(DOTA-3G-RGD2) has the tumor uptake in the range of 8.0 – 15 %ID/g with the tumor size being 0.05 – 0.30 g. When the tumor size is in the range of 0.5 – 0.6 g, the tumor uptake of 111In(DOTA-3G-RGD2)is only ~6.0 %ID/g.

Figure 4.

Figure 4

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The relationship between the tumor size and tumor uptake of 111In(DOTA-3G-RGD2) at 60 min p.i. in the athymic nude mice (n = 12) bearing the U87MG glioma xenografts.

Integrin αvβ3 Specificity

Figure 5 compares the 60-min organ uptake of 111In(DOTA-6G-RGD4) in the absence/presence of RGD2. Clearly, co-injection of RGD2 significantly blocked the tumor uptake of 111In(DOTA-6G-RGD4) (1.23 ± 0.26 %ID/g with RGD2 vs. 11.45 ± 2.36 %ID/g without RGD2 at 1 h p.i.). Its uptake in normal organs was also significantly blocked by co-injection of excess RGD2. For example, the uptake of 111In(DOTA-6G-RGD4) in eyes, heart, intestine, liver, lungs, and spleen was 1.54 ± 0.22, 1.49 ± 0.29, 9.25 ± 1.27, 3.77 ± 0.79, 3.44 ± 1.86 and 2.38 ± 0.49 %ID/g, respectively, without RGD2, while its uptake in the same organs was only 0.33 ± 0.06, 0.66 ± 0.18, 0.79 ± 0.10, 0.79 ± 0.20, 2.06 ± 1.12, and 0.54 ± 0.09 %ID/g, respectively, in the presence of excess RGD2.

Figure 5.

Figure 5

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Comparison of the 60-min biodistribution data of 111In(DOTA-6G-RGD4) in athymic nude mice (n = 5) bearing U87MG glioma xenografts in the absence/presence of excess RGD2 to demonstrate its integrin αvβ3–specificity.

Planar Imaging

Figure 6 illustrates the planar images of the glioma-bearing mice administered with ~3.7 MBq of 111In(DOTA-6G-RGD4) (top) 111In(DOTA-3G-RGD2) (bottom) and at 1, 4, 24 and 72 h p.i. The tumors could be clearly visualized with excellent T/B contrast as early as 1 h p.i. in the glioma-bearing mice administered with 111In(DOTA-6G-RGD4) and 111In(DOTA-3G-RGD2). Both 111In(DOTA-6G-RGD4) and 111In(DOTA-3G-RGD2) had a very long tumor retention time (t1/2 > 30 h), which was consistent with the results obtained from biodistribution studies (Tables 2 and 3). In addition, we also observed that the tumor distribution of 111In(DOTA-3G-RGD2) is quite heterogeneous most likely due to the heterogeneity of integrin αvβ3 overexpressed on tumor tissues.

Figure 6.

Figure 6

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The whole-body planar images of the tumor-bearing mice administered with ~100 μCi of 111In(DOTA-6G-RGD4) (top) and 111In(DOTA-3G-RGD2) (bottom) at 1, 4, 24 and 72 h p.i.

Metabolism

Figure 7 shows representative radio-HPLC chromatograms of 111In(DOTA-6G-RGD4) and 111In(DOTA-3G-RGD2) in saline before injection, in urine at 30 min and 120 min p.i., and in feces at 120 min p.i. The percentage radioactivity recovery from the urine and feces samples was >95% for both radiotracers. Attempts to extract radioactivity from the liver and kidneys were not successful due to limited radioactivity accumulation in both organs. There was very little metabolite detected in either urine or feces samples over the 2 h period for 111In(DOTA-3G-RGD2). For 111In(DOTA-6G-RGD4), however, there was a significant metabolism during its excretion via both renal and hepatobiliary routes. Only ~25% of 111In(DOTA-6G-RGD4) remained intact in the feces sample.

Figure 7.

Figure 7

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Typical radio-HPLC chromatograms of 111In(DOTA-6G-RGD4) (left) and 111In(DOTA-3G-RGD2) (right) in saline before injection, in urine at 30 min and 120 min p.i., and in feces at 120 min p.i. Each mouse was administered with ~100 μCi radiotracer.

DISCUSSION

In this study, we have prepared 111In complexes of DOTA-6G-RGD4, DOTA-RGD4 and DOTA-3G-RGD2. It was found that the integrin αvβ3 binding affinity follows the order of DOTA-6G-RGD4 > DOTA-RGD4 ~ DOTA-3G-RGD2 ≫ c(RGDyK). Two factors contribute to the higher integrin αvβ3 binding affinity of DOTA-6G-RGD4, DOTA-RGD4 and DOTA-3G-RGD2 as compared to c(RGDyK). Previously, we have clearly demonstrated that 3G-RGD2 binds to the integrin αvβ3 in a bivalent fashion while RGD2 is only monovalent due to the short distance (6 bonds excluding K residues) between two RGD motifs (31, 33, 37). The fact that DOTA-RGD4 (IC50 = 1.5 ± 0.2 nM) and DOTA-3G-RGD2 (IC50 = 1.3 ± 0.2 nM) shared almost identical IC50 values strongly suggests that DOTA-RGD4 is bivalent in binding to integrin αvβ3. This conclusion is supported by their similar tumor uptake (Figure 3), and is completely consistent with the biodistribution data obtained for the 99mTc-labeled cyclic RGD peptides 3G-RGD2 and RGD4 (31, 37). Were DOTA-RGD4 tetravalent, it would have had significantly higher integrin αvβ3 binding affinity than DOTA-3G-RGD2 whereas 111In(DOTA-RGD4) would have had higher initial tumor uptake than 111In(DOTA-3G-RGD2).

DOTA-6G-RGD4 (IC50 = 0.4 ± 0.1 nM) has higher integrin αvβ3 binding affinity than DOTA-RGD4 (IC50 = 1.5 ± 0.2 nM). As a result, 111In(DOTA-6G-RGD4) has slightly higher initial tumor uptake (Figure 3) than 111In(DOTA-RGD4). Previously, we found that both bivalency and the “locally enhanced RGD concentration” contribute to the higher integrin αvβ3 binding affinity of 3G-RGD2 and 3P-RGD2 as compared to that of c(RGDfK) (31–37). We also found that the contribution from the bivalency to integrin αvβ3 binding affinity and radiotracer tumor uptake are much more significant than that from the “locally enhanced RGD concentration” (31, 32). For example, the tumor uptake of the 99mTc-labeled 3G-RGD2 was almost 3x higher than that of the 99mTc-labeled c(RGDfK) in the same model (31). Thus, we believe that the differences in the integrin αvβ3 binding affinity of DOTA-6G-RGD4 and DOTA-RGD4 and the initial tumor uptake of 111In(DOTA-6G-RGD4) and 111In(DOTA-RGD4) are most likely caused by the number of possibilities to achieve simultaneous integrin αvβ3 binding for 6G-RGD4 and RGD4. The concentration factor exists in both DOTA-6G-RGD4 and DOTA-RGD4. The difference is that there are only two possibilities for DOTA-RGD4 to achieve bivalency while any two of the four RGD motifs in DOTA-6G-RGD4 can achieve bivalency due to the longer distance between RGD motifs. If DOTA-6G-RGD4 were tetravalent or trivalent, it would have had much higher integrin αvβ3 binding affinity than DOTA-RGD4 whereas 111In(DOTA-6G-RGD4) would have shown much higher initial tumor uptake than 111In(DOTA-RGD4). This conclusion is supported by the fact that 111In(DOTA-6G-RGD4) and 111In(DOTA-3G-RGD2) share a similar initial tumor uptake (Figure 3: 12.66 ± 1.25 %ID/g and 10.19 ± 3.61 %ID/g, respectively) at 0.5 h p.i. Even though DOTA-6G-RGD4 and DOTA-RGD4 may not be tetravalent, the extra cyclic RGD motifs in DOTA-6G-RGD4 and DOTA-RGD4 contribute significantly to the higher tumor uptake of 111In(DOTA-6G-RGD4) and 111In(DOTA-RGD4) than that of 111In(DOTA-3G-RGD2) at 24 – 72 h p.i. (Figure 3). The concentration factor might also explain why 111In(DOTA-6G-RGD4) and 111In(DOTA-RGD4) had higher uptake than 111In(DOTA-3G-RGD2) (Figure 3) in the intestine, liver and kidneys, all of which are integrin αvβ3-positive (15, 16). From this point of view, we believe that 111In(DOTA-6G-RGD4) is most likely bivalent in binding to the integrin αvβ3. However, the IC50 values and ex-vivo biodistribution data cannot completely eliminate the possibility of trivalency or tetravalency for 111In(DOTA-6G-RGD4).

For diagnostic radiotracers, the tumor retention time may not be as critical as tumor uptake and T/B ratios. For the 90Y and 177Lu radiotracers, however, the longer tumor retention time will result in higher radiation dose to the integrin αvβ3-positive tumors during the same period of time. It must be noted that the ability of a multimeric RGD peptide to achieve bivalency also depends on the integrin αvβ3 density. If the tumor integrin αvβ3 density is high, the distance between two neighboring integrin αvβ3 sites will be short, and it is easier for the multimeric RGD peptide to achieve bivalency. If the integrin αvβ3 density is very low, the distance between two neighboring integrin αvβ3 sites will be long, and it might be more difficult for the same multimeric RGD peptide to achieve simultaneous integrin αvβ3 binding.

The integrin αvβ3-specificity of 111In(DOTA-6G-RGD4) is demonstrated by the “blocking experiment” (Figure 5). The blockage of radiotracer uptake strongly suggests that its glioma localization is indeed integrin αvβ3-mediated. The partial uptake blockage in the eyes, heart, intestine, lungs, liver and spleen indicates that the accumulation of 111In(DOTA-6G-RGD4) in these organs is in large part integrin αvβ3-mediated. This conclusion is supported by the immunohistopathological studies (15, 16), which showed a strong positive staining of endothelial cells of small glomeruli vessels in kidneys and weak staining in branches of the hepatic portal vein. Similar results were also obtained for radiolabeled (99mTc and 64Cu) cyclic RGD peptide dimers, such as 3G-RGD2 and 3P-RGD2 (31–33, 36, 37).

It is well established that multimerization of cyclic RGD peptides can significantly improve the integrin αvβ3-targeting capability as evidenced by their higher integrin αvβ3 binding affinity and better tumor uptake with better tumor uptake than their monomeric counterparts (26, 30–37). However, this also leads to more radioactivity accumulation of radiolabeled multimeric cyclic RGD peptides in normal organs, as evidenced by the higher uptake of 111In(DOTA-6G-RGD4) and 111In(DOTA-RGD4) than 111In(DOTA-3G-RGD2) in the intestine, liver and kidneys. As a result, the tumor/liver and tumor/kidney ratios of 111In(DOTA-6G-RGD4) and 111In(DOTA-RGD4) are significantly (p < 0.01) lower than those of 111In(DOTA-3G-RGD2) at >1 h p.i. This is consistent with more accumulation in the abdominal region of tumor-bearing mice administered with 111In(DOTA-6G-RGD4) (Figure 6: top) than that with 111In(DOTA-3G-RGD2) (Figure 6: bottom). Thus, 111In(DOTA-3G-RGD2) is a better radiotracer than 111In(DOTA-6G-RGD4) and 111In(DOTA-RGD4) for diagnostic purposes. For the integrin αvβ3-targeted therapeutic radiotracers, however, 6G-RGD4 has advantages over RGD4 and 3G-RGD2 because 111In(DOTA-6G-RGD4) has a longer tumor retention time than both 111In(DOTA-3G-RGD2) and 111In(DOTA-RGD4). At this moment, it is not clear why 111In(DOTA-3G-RGD2) is able to maintain its integrity during its excretion from both renal and hepatobiliary routes while 111In(DOTA-6G-RGD4) has significant degradation during its excretion from the hepatobiliary route (Figure 7). It is likely that the metabolic instability of 111In(DOTA-6G-RGD4) during its hepatobiliary excretion is caused by its much larger size (M.W. = 4,269 Daltons) than that of 111In(DOTA-3G-RGD2) (M.W. = 2,326 Daltons).

The %ID tumor uptake reflects the total integrin αvβ3 expression level on tumor cells and tumor neovasculature while the %ID/g tumor uptake reflects the integrin αvβ3 density. When tumor is small (<0.05 g or 50 m3), there is little tumor-mass and angiogenesis. As a result, 111In(DOTA-3G-RGD2) has low %ID tumor uptake (Figure 4A). As tumor grows, the integrin αvβ3 level becomes higher, and the %ID tumor uptake of 111In(DOTA-3G-RGD2) increases (Figure 4A). However, the microvessel density decreases due to the maturation of blood vessels, and the integrin αvβ3 density also decreases due to larger interstitial space and higher collagen concentrations (38). In addition, parts of the tumor may become necrotic, leading to lower integrin αvβ3 density. It is not surprising that 111In(DOTA-3G-RGD2) had significantly less %ID/g uptake in larger tumors (Figure 4B). Similar results were also reported for 64Cu(DOTA-3P-RGD2) (33). The linear relationship between the tumor size and %ID tumor uptake suggests that 111In(DOTA-3G-RGD2) might have the potential for monitoring tumor growth or shrinkage. However, the relationship between the radiotracer uptake and integrin αvβ3 levels needs to be further established in our future studies. The ability to non-invasively quantify integrin αvβ3 level will provide a useful tool to select the patients more appropriately for anti-angiogenic therapy (39, 40).

CONCLUSION

In this study, we evaluated 111In(DOTA-6G-RGD4), 111In(DOTA-RGD4) and 111In(DOTA-3G-RGD2) for their potential as new integrin αvβ3-targeted radiotracers. On the basis of results from the integrin αvβ3 binding assay and biodistribution studies, it is concluded that (1) tetrameric RGD peptides 6G-RGD4 and RGD4 are not tetravalent; (2) 6G-RGD4, RGD4 and 3G-RGD2 most likely bind to the integrin αvβ3 in a bivalent fashion; (3) two extra cyclic RGD motifs in 6G-RGD4 and RGD4 contribute significantly to the longer tumor retention times of 111In(DOTA-6G-RGD4) and 111In(DOTA-RGD4) than that of 111In(DOTA-3G-RGD2); (4) further increase of multiplicity from cyclic RGD dimers, such as 3G-RGD2, has a adverse impact on T/B ratios due to the increased radioactivity accumulation in normal organs; and (5) the combination of high tumor uptake and prolonged tumor retention of 111In(DOTA-6G-RGD4) suggests that its 90Y and 177Lu analogs, M(DOTA-6G-RGD4) (M = 90Y and 177Lu), might have to potential for the targeted radiotherapy of glioblastoma.

Acknowledgments

This work is supported, in part, by Purdue University and research grants: R01 CA115883-A2 from National Cancer Institute (NCI), R21 HL08396-01 from National Heart, Lung, and Blood Institute (NHLBI), and DE-FG02-08ER64684 from the Department of Energy.

References

  • 1.Weber WA, Haubner R, Vabuliene E, Kuhnast B, Wester HJ, Schwaiger M. Tumor angiogenesis targeting using imaging agents. Q J Nucl Med. 2001;45:179–182. [PubMed] [Google Scholar]
  • 2.Costouros NG, Diehn FE, Libutti SK. Molecular imaging of tumor angiogenesis. J Cell Biochem Suppl. 2002;39:72–78. doi: 10.1002/jcb.10426. [DOI] [PubMed] [Google Scholar]
  • 3.Liu S, Robinson SP, Edwards DS. Integrin αvβ3 directed radiopharmaceuticals for tumor imaging. Drugs of Future. 2003;28:551–564. [Google Scholar]
  • 4.Haubner R, Wester HJ. Radiolabeled tracers for imaging of tumor angiogenesis and evaluation of antiantiogenic therapies. Curr Pharm Des. 2004;10:1439–1455. doi: 10.2174/1381612043384745. [DOI] [PubMed] [Google Scholar]
  • 5.Liu S. Radiolabeled multimeric cyclic RGD peptides as integrin αvβ3-targeted radiotracers for tumor imaging. Mol Pharm. 2006;3:472–487. doi: 10.1021/mp060049x. [DOI] [PubMed] [Google Scholar]
  • 6.Chen X. Multimodality imaging of tumor integrin αvβ3 expression. Mini-Rev Med Chem. 2006;6:227–234. doi: 10.2174/138955706775475975. [DOI] [PubMed] [Google Scholar]
  • 7.Cai W, Chen X. Multimodality Molecular imaging of tumor angiogenesis. J Nucl Med. 2008;49:113S–128S. doi: 10.2967/jnumed.107.045922. [DOI] [PubMed] [Google Scholar]
  • 8.Cai W, Niu G, Chen X. Imaging of integrins as biomarkers for tumor angiogenesis. Curr Pharmaceut Des. 2008;14:2943–2973. doi: 10.2174/138161208786404308. [DOI] [PubMed] [Google Scholar]
  • 9.Liu S. Radiolabeled cyclic RGD peptides as integrin αvβ3-targeted radiotracers: Maximizing Binding Affinity via Bivalency. Bioconjugate Chem (ASAP) 2009 doi: 10.1021/bc900167c. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Thumshirn G, Hersel U, Goodman SL, Kessler H. Multimeric cyclic RGD peptides as potential tools for tumor targeting: solid-phase peptide synthesis and chemoselective oxime ligation. Chem Eur J. 2003;9:2717–2725. doi: 10.1002/chem.200204304. [DOI] [PubMed] [Google Scholar]
  • 11.Poethko T, Schottelius M, Thumshirn G, Herz M, Haubner R, Henriksen G, Kessler H, Schwaiger M, Wester HJ. Chemoselective pre-conjugate radiohalogenation of unprotected mono- and multimeric peptides via oxime formation. Radiochimica Acta. 2004;92:317–327. [Google Scholar]
  • 12.Chen X, Liu S, Hou Y, Tohme M, Park R, Bading JR, Conti PS. MicroPET imaging of breast cancer αv-integrin expression with 64Cu-labeled dimeric RGD peptides. Mol Imag Biol. 2004;6:350–359. doi: 10.1016/j.mibio.2004.06.004. [DOI] [PubMed] [Google Scholar]
  • 13.Chen X, Tohme M, Park R, Hou Y, Bading JR, Conti PS. MicroPET imaging of breast cancer αv-integrin expression with 18F-labeled dimeric RGD peptide. Mol Imaging. 2004;3:96–104. doi: 10.1162/15353500200404109. [DOI] [PubMed] [Google Scholar]
  • 14.Wu Y, Zhang X, Xiong Z, Cheng Z, Fisher DR, Liu S, Gambhir SS, Chen X. MicroPET imaging of glioma integrin αvβ3 expression using 64Cu-labeled tetrameric RGD peptide. J Nucl Med. 2005;46:1707–1718. [PubMed] [Google Scholar]
  • 15.Zhang X, Xiong Z, Wu Y, Cai W, Tseng JR, Gambhir SS, Chen X. Quantitative PET imaging of tumor integrin αvβ3 expression with 18F-FRGD2. J Nucl Med. 2006;47:113–121. [PMC free article] [PubMed] [Google Scholar]
  • 16.Wu Z, Li Z, Chen K, Cai W, He L, Chin FT, Li F, Chen X. Micro-PET of tumor integrin αvβ3 expression using 18F-labeled PEGylated tetrameric RGD peptide (18F-FPRGD4) J Nucl Med. 2007;48:1536–1544. doi: 10.2967/jnumed.107.040816. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Li ZB, Chen K, Chen X. 68Ga-labeled multimeric RGD peptides for microPET imaging of integrin αvβ3 expression. Eur J Nucl Med Mol Imaging. 2008;35:1100–1108. doi: 10.1007/s00259-007-0692-y. [DOI] [PubMed] [Google Scholar]
  • 18.Liu S, Edwards DS, Ziegler MC, Harris AR, Hemingway SJ, Barrett JA. 99mTc-Labeling of a hydrazinonicotinamide-conjugated vitronectin receptor antagonist useful for imaging tumor. Bioconjugate Chem. 2001;12:624–629. doi: 10.1021/bc010012p. [DOI] [PubMed] [Google Scholar]
  • 19.Liu S, Cheung E, Ziegler MC, Rajopadhye M, Edwards DS. 90Y- and 177Lu-labeling of a DOTA-conjugated vitronectin receptor antagonist for tumor therapy. Bioconjugate Chem. 2001;12:559–568. doi: 10.1021/bc000146n. [DOI] [PubMed] [Google Scholar]
  • 20.Janssen M, Oyen WJG, Massuger LFAG, Frielink C, Dijkgraaf I, Edwards DS, Radjopadhye M, Corstens FH, Boerman OC. Comparison of a monomeric and dimeric radiolabeled RGD-peptide for tumor targeting. Cancer Biother Radiopharm. 2002;17:641–646. doi: 10.1089/108497802320970244. [DOI] [PubMed] [Google Scholar]
  • 21.Janssen M, Oyen WJG, Dijkgraaf I, Massuger LF, Frielink C, Edwards DS, Rajopadhye M, Boonstra H, Corstens FH, Boerman OC. Tumor targeting with radiolabeled integrin αvβ3 binding peptides in a nude mice model. Cancer Res. 2002;62:6146–6151. [PubMed] [Google Scholar]
  • 22.Liu S, Hsieh WY, Kim YS, Mohammed SI. Effect of coligands on biodistribution characteristics of ternary ligand 99mTc complexes of a HYNIC-conjugated cyclic RGDfK dimer. Bioconjuage Chem. 2005;16:1580–1588. doi: 10.1021/bc0501653. [DOI] [PubMed] [Google Scholar]
  • 23.Jia B, Shi J, Yang Z, Xu B, Liu Z, Zhao H, Liu S, Wang F. 99mTc-labeled cyclic RGDfK dimer: initial evaluation for SPECT imaging of glioma integrin αvβ3 expression. Bioconjugate Chem. 2006;17:1069–1076. doi: 10.1021/bc060055b. [DOI] [PubMed] [Google Scholar]
  • 24.Liu S, He ZJ, Hsieh WY, Kim YS, Jiang Y. Impact of PKM linkers on biodistribution characteristics of the 99mTc-labeled cyclic RGDfK dimer. Bioconjugate Chem. 2006;17:1499–1507. doi: 10.1021/bc060235l. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Liu S, Hsieh WY, Jiang Y, Kim YS, Sreerama SG, Chen X, Jia B, Wang F. Evaluation of a 99mTc-labeled cyclic RGD tetramer for noninvasive imaging integrin αvβ3-positive breast cancer. Bioconjugate Chem. 2007;18:438–446. doi: 10.1021/bc0603081. [DOI] [PubMed] [Google Scholar]
  • 26.Dijkgraaf I, Kruijtzer JAW, Liu S, Soede A, Oyen WJG, Corstens FHM, Liskamp RMJ, Boerman OC. Improved targeting of the αvβ3 integrin by multimerization of RGD peptides. Eur J Nucl Med Mol Imaging. 2007;34:267–273. doi: 10.1007/s00259-006-0180-9. [DOI] [PubMed] [Google Scholar]
  • 27.Dijkgraaf I, Liu S, Kruijtzer JAW, Soede AC, Oyen WJG, Liskamp RMJ, Corstens FH, Boerman OC. Effect of linker variation on the in vitro and in vivo characteristics of an 111In-labeled RGD Peptide. Nucl Med Biol. 2007;34:29–35. doi: 10.1016/j.nucmedbio.2006.10.006. [DOI] [PubMed] [Google Scholar]
  • 28.Jia B, Liu Z, Shi J, Yu ZL, Yang Z, Zhao HY, He Z, Liu S, Wang F. Linker effects on biological properties of 111In-labeled DTPA conjugates of a cyclic RGDfK dimer. Bioconjugate Chem. 2008;19:201–210. doi: 10.1021/bc7002988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Wang JJ, Kim YS, Liu S. 99mTc-labeling of HYNIC-conjugated cyclic RGDfK dimer and tetramer using EDDA as coligand. Bioconjugate Chem. 2008;19:634–642. doi: 10.1021/bc7004208. [DOI] [PubMed] [Google Scholar]
  • 30.Liu S, Kim YS, Hsieh WY, Sreerama SG. Coligand effects on solution stability, biodistribution and metabolism of 99mTc-labeled cyclic RGDfK tetramer. Nucl Med Biol. 2008;35:111–121. doi: 10.1016/j.nucmedbio.2007.08.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Shi J, Wang L, Kim YS, Zhai S, Liu Z, Chen X, Liu S. Improving tumor uptake and excretion kinetics of 99mTc-labeled cyclic Arginine-Glycine-Aspartic (RGD) dimers with triglycine linkers. J Med Chem. 2008;51:7980–7990. doi: 10.1021/jm801134k. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Wang L, Shi J, Kim YS, Zhai S, Jia B, Liu Z, Zhao H, Liu Z, Wang F, Chen X, Liu S. Improving tumor targeting capability and pharmacokinetics of 99mTc-labeled cyclic RGD dimers with PEG4 linkers. Mol Pharm. 2009;6:231–245. doi: 10.1021/mp800150r. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Shi J, Kim YS, Zhai S, Liu Z, Chen X, Liu S. Improving tumor uptake and pharmacokinetics of 64Cu-labeled cyclic RGD dimers with G3 and PEG4 Linkers. Bioconjugate Chem. 2009;20:750–759. doi: 10.1021/bc800455p. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Liu Z, Niu G, Shi J, Liu SL, Wang F, Liu S. 68Ga-Labeled cyclic RGD dimers with Gly3 and PEG4 linkers: promising agents for tumor integrin αvβ3 PET imaging. Eur J Nucl Med Mol Imaging. 2009;36:947–957. doi: 10.1007/s00259-008-1045-1. [DOI] [PubMed] [Google Scholar]
  • 35.Liu Z, Liu S, Wang F, Liu S, Chen X. Non-invasive imaging of tumor integrin expression using 18F-labeled RGD dimer peptide with PEG4 linkers. Eur J Nucl Med Mol Imaging. 2009;36:1296–1307. doi: 10.1007/s00259-009-1112-2. [DOI] [PubMed] [Google Scholar]
  • 36.Shi J, Kim YS, Chakraborty S, Jia B, Wang F, Liu S. 2-Mercaptoacetylglycylglycyl (MAG2) as a bifunctional chelator for 99mTc-labeling of cyclic RGD dimers: effects of technetium chelate on tumor uptake and pharmacokinetics. Bioconjugate Chem. 2009;20:1559–1568. doi: 10.1021/bc9001739. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Shi J, Wang L, Kim YS, Zhai S, Jia B, Zhao H, Wang F, Liu S. 99mTcO(MAG2-3G3-dimer): a new integrin αvβ3-targeted SPECT radiotracer with high tumor uptake and favorable pharmacokinetics. Eur J Nucl Med Mol Imaging. 2009;36:1874–1884. doi: 10.1007/s00259-009-1166-1. [DOI] [PubMed] [Google Scholar]
  • 38.Jain RK. Transport of molecules in the tumor interstitium: a review. Cancer Res. 1987;47:3039–3051. [PubMed] [Google Scholar]
  • 39.Cai W, Rao J, Gambhir SS, Chen X. How molecular imaging is speeding up antiangiogenic drug development. Mol Cancer Ther. 2006;5:2624–2633. doi: 10.1158/1535-7163.MCT-06-0395. [DOI] [PubMed] [Google Scholar]
  • 40.Niu G, Chen X. Has molecular and cellular imaging enhanced drug discovery and drug development? Drugs in R&D. 2008;9:351–368. doi: 10.2165/0126839-200809060-00002. [DOI] [PubMed] [Google Scholar]

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