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Theranostics 2011; 1:322-340. doi:10.7150/thno/v01p0322 This volume Cite

Research Paper

Evaluation of ¹¹¹In-Labeled Cyclic RGD Peptides: Effects of Peptide and Linker Multiplicity on Their Tumor Uptake, Excretion Kinetics and Metabolic Stability

Jiyun Shi^1,2, Yang Zhou¹, Sudipta Chakraborty¹, Young-Seung Kim¹, Bing Jia², Fan Wang², Shuang Liu¹

1. School of Health Sciences, Purdue University, IN 47907, USA
2. Medical Isotopes Research Center, Peking University, Beijing 100083, China

✉ Corresponding author: School of Health Sciences, Purdue University, 550 Stadium Mall Drive, West Lafayette, IN 47907. Phone: 765-494-0236; Fax 765-496-1377; Email: liu100edu

Abstract

Purpose: The purpose of this study was to demonstrate the valence of cyclic RGD peptides, P-RGD (PEG₄-c(RGDfK): PEG₄ = 15-amino-4,710,13-tetraoxapentadecanoic acid), P-RGD₂ (PEG₄-E[c(RGDfK)]₂, 2P-RGD₄ (E{PEG₄-E[c(RGDfK)]₂}₂, 2P4G-RGD₄ (E{PEG₄-E[G₃-c(RGDfK)]₂}₂: G₃ = Gly-Gly-Gly) and 6P-RGD₄ (E{PEG₄-E[PEG₄-c(RGDfK)]₂}₂) in binding to integrin α_vβ₃, and to assess the impact of peptide and linker multiplicity on biodistribution properties, excretion kinetics and metabolic stability of their corresponding ¹¹¹In radiotracers.

Methods: Five new RGD peptide conjugates (DOTA-P-RGD (DOTA =1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid), DOTA-P-RGD₂, DOTA-2P-RGD₄, DOTA-2P4G-RGD₄, DOTA-6P-RGD₄), and their ¹¹¹In complexes were prepared. The integrin α_vβ₃ binding affinity of cyclic RGD conjugates were determined by a competitive displacement assay against ¹²⁵I-c(RGDyK) bound to U87MG human glioma cells. Biodistribution, planar imaging and metabolism studies were performed in athymic nude mice bearing U87MG human glioma xenografts.

Results: The integrin α_vβ₃ binding affinity of RGD conjugates follows the order of: DOTA-6P-RGD₄ (IC₅₀ = 0.3 ± 0.1 nM) ~ DOTA-2P4G-RGD₄ (IC₅₀ = 0.2 ± 0.1 nM) ~ DOTA-2P-RGD₄ (IC₅₀ = 0.5 ± 0.1 nM) > DOTA-3P-RGD₂ (DOTA-PEG₄-E[PEG₄-c(RGDfK)]₂: IC₅₀ = 1.5 ± 0.2 nM) > DOTA-P-RGD₂ (IC₅₀ = 5.0 ± 1.0 nM) >> DOTA-P-RGD (IC₅₀ = 44.3 ± 3.5 nM) ~ c(RGDfK) (IC₅₀ = 49.9 ± 5.5 nM) >> DOTA-6P-RGK₄ (IC₅₀ = 437 ± 35 nM). The fact that DOTA-6P-RGK₄ had much lower integrin α_vβ₃ binding affinity than DOTA-6P-RGD₄ suggests that the binding of DOTA-6P-RGD₄ to integrin α_vβ₃ is RGD-specific. This conclusion is consistent with the lower tumor uptake for ¹¹¹In(DOTA-6P-RGK₄) than that for ¹¹¹In(DOTA-6P-RGD₄). It was also found that the G₃ and PEG₄ linkers between RGD motifs have a significant impact on the integrin α_vβ₃-targeting capability, biodistribution characteristics, excretion kinetics and metabolic stability of ¹¹¹In-labeled cyclic RGD peptides.

Conclusion: On the basis of their integrin α_vβ₃ binding affinity and tumor uptake of their corresponding ¹¹¹In radiotracers, it was conclude that 2P-RGD₄, 2P4G-RGD₄ and 6P-RGD₄ are most likely bivalent in binding to integrin α_vβ₃, and extra RGD motifs might contribute to the long tumor retention times of ¹¹¹In(DOTA-2P-RGD₄), ¹¹¹In(DOTA-2P4G-RGD₄) and ¹¹¹In(DOTA-6P-RGD₄) than that of ¹¹¹In(DOTA-3P-RGD₃) at 72 h p.i. Among the ¹¹¹In-labeled cyclic RGD tetramers evaluated in the glioma model, ¹¹¹In(DOTA-2P4G-RGD₄) has very high tumor uptake with the best tumor/kidney and tumor/liver ratios, suggesting that ⁹⁰Y(DOTA-2P4G-RGD₄) and ¹⁷⁷Lu(DOTA-2P4G-RGD₄) might have the potential for targeted radiotherapy of integrin α_vβ₃-positive tumors.

Keywords: integrin α_vβ₃, ¹¹¹In-labeled cyclic RGD peptides, tumor imaging

Introduction

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 develop the integrin α_vβ₃-targeted radiotracers for noninvasive imaging of rapidly growing and metastatic tumors [1-14]. Multiple cyclic RGD moieties are utilized to maximize their integrin α_vβ₃ binding affinity and the radiotracer tumor uptake [9, 15-46]. It was found that the radiolabeled (^99mTc, ¹⁸F, ⁶⁴Cu, ⁶⁸Ga and ¹¹¹In) cyclic RGD tetramers have better tumor-targeting capability as evidenced by their higher tumor uptake with longer tumor retention time than their corresponding dimeric and monomeric counterparts [20, 22, 29, 32, 34, 35, 44, 46]. It was also found that cyclic RGD tetramers, such as DOTA-RGD₄ (DOTA = 1,4,7,10-tetraazacyclodo­decane-1,4,7,10-tetracetic acid) and DOTA-E{G₃-E[G₃-c(RGDfK)]₂}₂ (Figure 1: DOTA-6G-RGD₂) are likely bivalent in binding to integrin α_vβ₃ even though they contain four identical RGD motifs [46]. The key for bivalency is the distance between two RGD motifs [36-38]. For example, RGD₂ was monovalent whereas 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) are bivalent because of the increased distance between two cyclic RGD motifs for simultaneous integrin α_vβ₃ binding [38, 45].

To further prove the “bivalency concept”, we prepared three new cyclic RGD peptide tetramers (Figure 1: 2P-RGD₄, 2P4G-RGD₄ and 6P-RGD₄). We were particularly interested in these three peptides because they contain different number of G₃ and PEG₄ linkers between cyclic RGD motifs. The main objective of this study was to demonstrate their valence in binding to integrin α_vβ₃, and to assess the impact of cyclic RGD peptide and linker multiplicity on biodistribution properties, excretion kinetics and metabolic stability of ¹¹¹In radiotracers. In this report, we present the synthesis and biological evaluation of RGD tetramer conjugates, DOTA-2P-RGD₄, DOTA-2P4G-RGD₄ and DOTA-6P-RGD₄, and their ¹¹¹In complexes. A whole-cell competitive displacement assay was used to determine their integrin α_vβ₃ binding affinities against ¹²⁵I-c(RGDyK) bound to U87MG human glioma cells. Biodistribution and planar imaging studies performed in the athymic nude mice bearing U87MG glioma xenografts. For comparison purposes, we prepared and evaluated conjugates DOTA-PEG₄-c(RGDfK) (DOTA-P-RGD), DOTA-PEG₄-E[c(RGDfK)]₂ (DOTA-P-RGD₂), and their ¹¹¹In radiotracers using the same in vitro and in vivo assays. In addition, we prepared a “scrambled” peptide conjugate DOTA-E{PEG₄-E[PEG₄-c(RGKfD)]₂}₂ (DOTA-6P-RGK₄), which has the same amino acid residues as those in DOTA-6P-RGD₄ using c(RGKfD) motifs instead of c(RGDfK), and ¹¹¹In(DOTA-6P-RGK₄) to demonstrate the RGD-specificity of ¹¹¹In radiotracers.

Materials and Methods

Materials and Instruments. Chemicals were purchased from Sigma/Aldrich (St. Louis, MO), and were used without purification. Cyclic peptides, PEG₄-c(RGDfK) (P-RGD), PEG₄-E[G₃-c(RGDfK)]₂ (P2G-RGD₂), PEG₄-E[PEG₄-c(RGDfK)]₂ (3P-RGD₂) and PEG₄-E[PEG₄-c(RGKfD)]₂ (3P-RGK₂), were all custom-made by 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). The Boc-protected glutamic acid N-hydroxysuccinamide ester (Boc-E(OSu)₂) and PEG₄-E[c(RGDfK)]₂ (P-RGD₂) were prepared according to the methods described in our previous reports [23-26]. Syntheses and biological evaluations of DOTA-3P-RGD₂ and ¹¹¹In(DOTA-3P-RGD₂) were reported in our previous communication [45]. ESI (electron-spray ionization) mass spectral data were obtained in positive mode using a Finnigan LCQ classic mass spectrometer, School of Pharmacy, Purdue University. The MALDI (matrix assisted laser desorption ionizations) mass spectral data were collected using an Applied Biosystems Voyager DE PRO mass spectrometer (Framingham, MA), the Department of Chemistry, Purdue University. ¹¹¹InCl₃ was obtained from Perkin-Elmer Life 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. The HPLC method (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 x 250 mm, 100 Å pore size; Agilent Technologies, Santa Clara, CA). The flow rate was 2.5 mL/min with the gradient mobile phase going from 90% A (0.1% TFA in water) and 10% (0.1% TFA in acetonitrile) at 0 min to 20% B at 5 min and to 40% B at 20 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 x 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 going from 10% B at 5 min to 20% B at 15 min and to 50% B at 20 min.

 Figure 1 

Cyclic RGD peptide tetramer conjugates: DOTA-6G-RGD₄, DOTA-6P-RGD₄, DOTA-2P-RGD₄ and DOTA-2P4G-RGD₄.

DOTA-PEG₄-c(RGDfK) (DOTA-P-RGD). PEG₄-c(RGDfK) (7.1 mg, ~8.3 μmol) and DOTA-OSu (5.5 mg, ~11.0 μmol) were dissolved in 1 mL of anhydrous dimethylformamide (DMF). The pH value in the reaction mixture was adjusted to 8.5 - 9.0 using diisopropylethylamine (DIEA). The mixture was then stirred overnight at room temperature. After addition of 3 mL of 100 mM NH₄OAc buffer (pH = 7.0), the resulting solution was subjected to HPLC-purification (Method 1). The fraction at ~ 12.5 min was collected. Lyophilization of collected fractions afforded conjugate DOTA-P-RGD. The yield was 4.1 mg (~40%). ESI-MS (positive mode): m/z =1237.58 for [M + H]⁺ (M =1236.6 calcd. for C₅₄H₈₈N₁₄O₁₉).

DOTA-PEG₄-[c(RGDfK)]₂ (DOTA-P-RGD₂). PEG₄-[c(RGDfK)]₂ (1.8 mg, 1.1 μmol) and DOTA-OSu (2.1 mg, 4.2 μmol) were dissolved in DMF (1 mL). The pH in the reaction mixture was adjusted to 8.5 - 9.0 using DIEA. The 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 ~ 16.4 min afforded 0.9 mg of DOTA-P-RGD₂ (~41%) with 95% HPLC purity. The yield was ESI-MS (positive mode) for m/z = 1951.5 for [M + H]⁺, 976.6 for [M + 2H]²⁺ and 1974.4 for [M + Na]⁺ (M = 1950.2 calcd. for [C₈₆H₁₃₄N₂₄O₂₈]⁺).

Boc-E{PEG₄-E[c(RGDfK)]₂}₂ (Boc-2P-RGD₄). To a solution of Boc-E(OSu)₂ (1.1 mg, 2.5 μmol) in 1 mL of DMF were added PEG₄-[c(RGDfK)]₂ (5.8 mg, 3.4 μmol) and DIEA (2 drops). The reaction mixture was stirred for 2 h at room temperature. The reaction was terminated by adding 3 mL NH₄OAc buffer (100 mM, pH = 7.0). The product was isolated by HPLC (Method 1). Lyophilization of collected fractions at 18.2 min afforded Boc-2P-RGD₄. The yield was 4.5 mg (~79%). ESI-MS (positive mode): m/z = 3344.2 for [M + H]⁺ and 1671.9 for [M + 2H]²⁺ (M = 3342.7 calcd. for [C₁₅₀H₂₂₉N₄₁O₄₆]⁺).

E{PEG₄-E[c(RGDfK)]₂}₂ (2P-RGD₄). The Boc-2P-RGD₄ (4.3 mg, 1.3 μmol) was dissolved in 2 mL of TFA. After standing at room temperature for 5 min, 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 by HPLC (Method 1). Lyophilization of collected fractions at 15.7 min afforded 2P-RGD₄. The yield was 2.6 mg (~59%). ESI-MS (positive mode): m/z = 3244.0 for [M + H]⁺ and 1620.5 for [M + 2H]²⁺ (M = 3242.6 calcd. for [C₁₄₅H₂₂₁N₄₁O₄₄]⁺).

DOTA-E{PEG₄-E[c(RGDfK)]₂}₂ (DOTA-2P-RGD₄). DOTA-OSu (3.3 mg, 6.5 μmol) and 2P-RGD₄ (2.2 mg, 0.7 μmol) were dissolved in 1 mL of anhydrous DMF. After addition of 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 isolated by HPLC (Method 1). Lyophilization of collected fraction at 16.1 min afforded conjugate DOTA-2P-RGD₄ with >95% HPLC purity. The yield was 1.4 mg (~56%). MALDI-MS: m/z = 3626.4 for [M + H]⁺ (M = 3626.8 calcd. for [C₁₆₁H₂₄₇N₄₅O₅₁]⁺).

Boc-E{PEG₄-E[Gly₃-c(RGDfK)]₂}₂ (Boc-2P4G-RGD₄). To a solution of Boc-E(OSu)₂ (3.8 mg, 8.6 μmol) in DMF (3 mL) were added P2G-RGD₂ (20.9 mg, 11.0 μmol) and excess DIEA (3 drops). The mixture was stirred for 3 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 collected fractions at 16.3 min afforded Boc-2P4G-RGD₄. The yield was 9.5 mg (~ 43 %). ESI-MS (positive mode): m/z = 4028.0 for [M + H]⁺ (M = 4027.3 calcd. for [C₁₇₄H₂₆₅N₅₃O₅₈]⁺).

E{PEG₄-E[Gly₃-c(RGDfK)]₂}₂ (2P4G-RGD₄). The Boc-protected 2P4G-RGD₄ (9.0 mg, 2.2 μmol) was dissolved in 2 mL of TFA. After standing at room temperature for 5 min, TFA was removed, and 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 collected fractions at 14.2 min afforded 2P4G-RGD₄. The yield was 4.0 mg (~46%). ESI-MS (positive mode): m/z = 3927.9 for [M + H]⁺ and 1964.6 for [M + 2H]²⁺ (M = 3927.3 calcd. for [C₁₆₉H₂₅₇N₅₃O₅₆]⁺).

DOTA-E{PEG₄-E[Gly₃-c(RGDfK)]₂}₂ (DOTA-2P4G-RGD₄). DOTA-OSu (5.8 mg, 11.5 μmol) and 2P4G-RGD₄ (3.2 mg, 0.8 μmol) were dissolved in DMF (2 mL). After addition of DIEA (2 drops), the 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 collected fraction at 14.9 min afforded conjugate DOTA-2P4G-RGD₄ with >95% HPLC purity. The yield was 2.2 mg (~64%). MALDI-MS: m/z = 4315.4 for [M + H]⁺ (M = 4313.6 calcd. for [C₁₈₅H₂₈₃N₅₇O₆₃]⁺).

Boc-E{PEG₄-E[PEG₄-c(RGDfK)]₂}₂ (Boc-6P-RGD₄). To a solution of Boc-E(OSu)₂ (2.1 mg, 4.75 μmol) in DMF (2 mL) was added 3P-RGD₂ (22 mg, 10.7 μmol). After addition of excess DIEA, the reaction mixture was stirred for 3 h at room temperature. The reaction was terminated by addition of 3 mL NH₄OAc buffer (100 mM, pH = 7.0). The product was separated by HPLC (Method 1). Lyophilization of collected fractions at 18.5 min afforded desired product Boc-6P-RGD₄. The yield was 15.6 mg (~76%). MALDI-MS: m/z = 4333.8 for [M + H]⁺ (M = 4331.8 calcd. for [C₁₉₄H₃₁₃N₄₅O₆₆]⁺).

E{PEG₄-E[PEG₄-c(RGDfK)]₂}₂ (6P-RGD₄). The Boc-protected 6P-RGD₄ (15.6 mg, 3.6 μmol) was dissolved in 2 mL of TFA. After standing 5 min at room temperature, TFA was removed, and the residue was dissolved in 2 mL of NH₄OAc buffer (100 mM, pH = 7.0). The product was separated from the mixture by HPLC (Method 1). Lyophilization of collected fractions at 17.3 min afforded the desired product 6P-RGD₄. The yield was 14.0 mg (~92%). MALDI-MS: m/z = 4233.4 for [M + H]⁺ (M = 4231.7 calcd. for [C₁₈₉H₃₀₅N₄₅O₆₄]⁺).

DOTA-E{PEG₄-E[PEG₄-c(RGDfK)]₂}₂ (DOTA-6P-RGD₄). DOTA-OSu (7.7 mg, 15.3 μmol) and 6P-RGD₄ (3.8 mg, 0.9 μmol) were dissolved in 1 mL of anhydrous DMF. After addition of 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 from the mixture by HPLC (Method 1). Lyophilization of collected fraction at 18.2 min afforded DOTA-6P-RGD₄ with >95% purity. The yield was 1.8 mg (~43%). MALDI-MS: m/z = 4622.7 for [M + H]⁺ (M = 4618.1 calcd. for [C₂₀₅H₃₃₁N₄₉O₇₁]⁺).

Boc-E{PEG₄-E[PEG₄-c(RGKfD)]₂}₂ (Boc-6P-RGK₄). To a solution of the Boc-E(OSu)₂ (5.5 mg, 12.5 μmol) in DMF (2 mL) was added 3P-RGK₂ (30 mg, 14.6 μmol). After addition of excess DIEA, the reaction mixture was stirred for 3 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 collected fractions at 18.5 min afforded desired product Boc-6P-RGK₄. The yield was 15.6 mg (~49%). MALDI-MS: m/z = 4334.9 for [M + H]⁺ (M = 4331.8 calcd. for [C₁₉₄H₃₁₃N₄₅O₆₆]⁺).

E{PEG₄-E[PEG₄-c(RGKfD)]₂}₂ (6P-RGK₄). Boc-6P-RGK₄ (15.5 mg, 3.6 μmol) was dissolved in TFA (2 mL). After 5 min at room temperature, TFA was removed, and the residue was dissolved in 2 mL of NH₄OAc buffer (100 mM, pH = 7.0). The product was separated from the mixture by HPLC (Method 1). Lyophilization of collected fractions at 15.8 min afforded 6P-RGK₄. The yield was 14.0 mg (~93%). MALDI-MS: m/z = 4233.4 for [M + H]⁺ (M = 4231.7 calcd. for [C₁₈₉H₃₀₅N₄₅O₆₄]⁺).

DOTA-E{PEG₄-E[PEG₄-c(RGKfD)]₂}₂ (DOTA-6P-RGK₄). DOTA-OSu (5.0 mg, 10.0 μmol) and 6P-RGK₄ (3.8 mg, 0.9 μmol) were dissolved in DMF (1 mL). After addition of 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 from the mixture by HPLC (Method 1). Lyophilization of the collected fraction at ~18.6 min afforded DOTA-6P-RGK₄ with >95% HPLC purity. The yield was 2.8 mg (~68%). MALDI-MS: m/z = 4622.7 for [M + H]⁺ (M = 4618.1 calcd. for [C₂₀₅H₃₃₁N₄₉O₇₁]⁺).

¹¹¹In-Labeling. To a clean Eppendorf tube were added 200 μL of the DOTA conjugate solution (2.5 mg/mL in 0.1 M NaOAc buffer, pH = 5.5) and 20 μL of ¹¹¹InCl₃ solution (~500 μCi in 0.05 M HCl). The vial was heated in a water bath at 100 ^oC for ~ 15 min. After cooling to room temperature, a sample of the resulting solution was analyzed by radio-HPLC (Method 2). The water-octanol partition coefficients of all ¹¹¹In radiotracers were determined according to literature methods [40, 41]. The log P values were reported as an average of three independent measurements plus the standard deviation.

Dose Preparation. For biodistribution studies, all new ¹¹¹In radiotracers were purified by HPLC (Method 2). Volatiles in the HPLC mobile phase were removed under vacuum (~5 mmHg) at room temperature. Doses were prepared by dissolving the ¹¹¹In radiotracer in saline to 70 - 100 μCi/mL. For the blocking experiment, excess RGD₂ was dissolved in the dose solution to 7.5 mg/mL. For planar imaging studies, doses were prepared by dissolving the radiotracer (no purification) in saline to ~1 mCi/mL. In all cases, the dose solution was filtered with a 0.20 μm Millex-LG filter before being injected into the animals. Each animal was injected with ~0.1 mL of the dose solution.

In Vitro Whole-Cell Integrin α_vβ₃ Binding Assay. The integrin binding affinity and specificity of cyclic 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^TM (GraphPad Software, Inc., San Diego, CA). Experiments were carried out twice with triplicate samples. 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 with NIH animal experiment guidelines (Principles of Laboratory Animal Care, NIH Publication No. 86-23, revised 1985). The animal protocol has been 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 U87MG 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 were performed in a laminar flow cabinet using aseptic technique. Four weeks after inoculation, the tumor size was 0.1 - 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 ~3 μCi 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 tumor-bearing mice. The 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 Perkin Elmer Wizard - 1480 γ-counter (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 ~3 μCi of ¹¹¹In(DOTA-6P-RGD₄) along with ~350 μg (~14 mg/kg) of RGD₂. Such a high dose of RGD₂ was used to make sure that the integrin α_vβ₃ 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 deviation 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.

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

Western Blot. The xenografted U87MG glioma tumor tissue and normal organ tissues (lung, liver and kidney) were excised and homogenized in the RIPA buffer. Protease inhibitors were added freshly into the lysis buffer before experiments. After sonication, lysates were centrifuged at 12,000 g at 4 °C for 15 min. The protein concentration was determined using the Bio-Rad Protein Assay Reagent (Bio-Rad Laboratories Inc., Hercules, CA). The protein lysates (50 μg) were separated by 10% SDS-PAGE and transferred to PVDF membranes for detection of integrin β₃. The blots were incubated in blocking solution consisting of 5% milk and 3% BSA in TBS-T for 1 h at room temperature and then were immunoblotted at room temperature for 2 h with the rabbit polyclonal anti-β3 primary antibody (1:1000, Cell Signaling Technology, Danvers, MA). The anti-rabbit secondary antibody was incubated with the blots for another 1 h at room temperature. Detection by enzyme-linked chemiluminescence was performed according to the manufacturer's protocol (Thermo Fisher Scientific, Rockford, IL).

Planar Imaging. The imaging studies were performed on ¹¹¹In(DOTA-2P-RGD₄), ¹¹¹In(DOTA-2P4G-RGD₄) and ¹¹¹In(DOTA-6P-RGD₄) using the athymic nude mice (n = 3) bearing U87MG human glioma xenografts. Each tumor-bearing mouse was administered with ~100 μCi of ¹¹¹In radiotracer in 0.1 mL saline 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 gamma camera (Diagnostic Services Inc., NJ) equipped with a parallel-hole, medium-energy and high-resolution collimator. The whole-body anterior images were acquired at 0.5, 1, 4, 24 and 72 h p.i. and stored digitally in a 128 x 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. Athymic nude mice were used for metabolism studies. Each mouse was administered with ~100 μCi of ¹¹¹In(DOTA-2P-RGD₄), ¹¹¹In(DOTA-2P4G-RGD₄) and ¹¹¹In(DOTA-6P-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 for 5 min at room temperature. The supernatant was collected and passed through a 0.20 micron Millex-LG filter unit to remove any precipitate or particles. The filtrate was analyzed by radio-HPLC (Method 2). 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 μm Millex-LG filter unit. The filtrate was analyzed by radio-HPLC (Method 2). The 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 then homogenized. The homogenate was mixed with 2 mL 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. 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). The percentage of radioactivity recovery was >95% (by γ-counting) for both urine and feces samples.

Results

Cyclic RGD Conjugates. Syntheses of DOTA-P-RGD and DOTA-P-RGD₂ were straightforward by conjugation of DOTA-OSu with PEG₄-c(RGDfK) and PEG₄-E[c(RGDfK)]₂, respectively, in DMF in the presence of excess DIEA. The key step for successful preparation of DOTA-2P-RGD₄, DOTA-2P4G-RGD₄ and DOTA-6P-RGD₄ was the deprotection of Boc-2P-RGD₄, Boc-2P4G-RGD₄ and Boc-6P-RGD₄, respectively. In general, excess TFA must be removed within 10 min of the reaction in order to minimize formation of hydrolyzed products. Conjugation of 2P-RGD₄, 2P4G-RGD₄ and 6P-RGD₄ with DOTA-OSu in anhydrous DMF afforded their corresponding conjugates DOTA-2P-RGD₄, DOTA-2P4G-RGD₄ and DOTA-6P-RGD₄, respectively. DOTA-6P-RGK₄ was prepared using the identical procedure to that for DOTA-6P-RGD₄. DOTA-6P-RGK₄ has the identical amino acids, but their sequence was scrambled to demonstrate the RGD-specificity of DOTA-6P-RGD₄. The identities for all new RGD conjugates were confirmed by ESI-MS (positive mode) or MALDI-MS. Their HPLC purity was >95% before being used for ¹¹¹In-labeling and determination of their integrin α_vβ₃ binding affinity.

Integrin α_vβ₃ Binding Affinity. Figure 2 shows the displacement curves of ¹²⁵I-c(RGDyK) bound to U87MG glioma cells in the presence of cyclic RGD peptides. Their IC₅₀ values were obtained from curve fitting from Figure 2, and were calculated to be 49.9 ± 5.5, 44.3 ± 3.5, 5.0 ± 1.0, 1.5 ± 0.2, 0.5 ± 0.1, 0.2 ± 0.1, 0.3 ± 0.1, and 437 ± 35 nM for c(RGDfK), DOTA-P-RGD, DOTA-P-RGD₂, DOTA-3P-RGD₂, DOTA-2P-RGD₄, DOTA-2P4G-RGD₄, DOTA-6P-RGD₄ and DOTA-6P-RGK₄, respectively. DOTA-6P-RGK₄ has much lower binding affinity than DOTA-6P-RGD₄ (Figure 2). The IC₅₀ of DOTA-3P-RGD₂ was almost identical to that (IC₅₀ = 1.3 ± 0.2 nM) presented in our previous report [45].

Radiochemistry. All ¹¹¹In radiotracers were prepared by reacting ¹¹¹InCl₃ with the respective DOTA conjugates (DOTA-P-RGD, DOTA-P-RGD₂, DOTA-2P-RGD₄, DOTA-2P4G-RGD₄, DOTA-6P-RGD₄ and DOTA-6P-RGK₄) in NH₄OAc buffer (100 mM, pH = 5.5). Radiolabeling was completed by heating the reaction mixture at 100 ^oC for ~15 min. The radiochemical purity was >95% with the specific activity of > 40 mCi/μmol for all ¹¹¹In radiotracers. Their HPLC retention times and log P values were listed in Table 1. We found that all new ¹¹¹In radiotracers remain stable for more than 72 h in the kit matrix or after HPLC purification in the presence of 3 mM EDTA.

Biodistribution Characteristics. Selected biodistribution data for ¹¹¹In(L) (L = DOTA-P-RGD, DOTA-P-RGD₂, DOTA-2P-RGD₄, DOTA-2P4G-RGD₄, DOTA-6P-RGD₄ and DOTA-6P-RGK₄) are listed in Tables 2-6. Figure 3 compares their uptake (%ID/g) in the tumor and intestine, as well as their tumor/kidney and tumor/liver ratios in athymic nude mice (n = 5) bearing U87MG glioma xenografts. The biodistribution data and T/B ratios for ¹¹¹In(DOTA-3P-RGD₃) and ¹¹¹In(DOTA-6G-RGD₄) were obtained from our previous reports [45, 46]. The comparison of their tumor uptake will allow us to determine if the cyclic RGD tetramers (DOTA-2P-RGD₄, DOTA-2P4G-RGD₄ and DOTA-6P-RGD₄) are actually tetravalent in binding to integrin α_vβ₃.

 Figure 2 

Competitive inhibition curves of ¹²⁵I-c(RGDyK) bound to the U87MG human glioma cells by c(RGDyK), DOTA-P-RGD, DOTA-P-RGD₂, DOTA-3P-RGD₂, DOTA-2P-RGD₄, DOTA-2P4G-RGD₄, DOTA-6P-RGD₄ and DOTA-6P-RGK₄. Their IC₅₀ values were calculated to be 49.9 ± 5.5, 44.3 ± 3.5, 5.0 ± 1.0, 1.5 ± 0.2, 0.5 ± 0.1, 0.2 ± 0.1, 0.3 ± 0.1, and 437 ± 35 nM, respectively. The integrin α_vβ₃ binding affinity follows the order: DOTA-6P-RGD₄ ~ DOTA-2P4G-RGD₄ ~ DOTA-2P-RGD₄ > DOTA-3P-RGD₂ > DOTA-P-RGD₂ > DOTA-P-RGD >> DOTA-6P-RGK₄.

 Figure 3 

Comparison of the uptake (%ID/g) in tumor and intestine, as well as the tumor/kidney and tumor/liver ratios for ¹¹¹In(L) (L = DOTA-P-RGD, DOTA-P-RGD₂, DOTA-3P-RGD₂, DOTA-2P-RGD₄, DOTA-2P4G-RGD₄, DOTA-6P-RGD₄ and DOTA-6G-RGD₄) in the athymic nude mice (n = 5) bearing U87MG glioma xenografts. Biodistribution data and T/B ratios for ¹¹¹In(DOTA-3P-RGD₃) and ¹¹¹In(DOTA-6G-RGD₄) were obtained from our previous reports [45, 46].

In the glioma model, ¹¹¹In(DOTA-6P-RGD₄) was excreted mainly via renal route with >75% of the injected radioactivity recovered from urine and <10% of the injected radioactivity from feces. ¹¹¹In(DOTA-6P-RGD₄) had a rapid blood clearance with high tumor/blood ratios (Table 6). It had a high initial tumor uptake (10.5 %ID/g at 0.5 h p.i.) with a slow tumor radioactivity washout (t_1/2 ~ 40 h). ¹¹¹In(DOTA-6P-RGD₄) had a moderately low liver uptake with high tumor/liver ratio (Table 6). However, ¹¹¹In(DOTA-6P-RGD₄) had significant uptake in kidneys and intestine. Similar results were also obtained for ¹¹¹In(DOTA-6G-RGD₄) in the same animal model [46]. ¹¹¹In(DOTA-2P4G-RGD₄) was also excreted through the renal route with >80% of the injected radioactivity recovered from urine samples. ¹¹¹In(DOTA-2P4G-RGD₄) had a rapid blood clearance with high tumor/blood ratios (Table 5). The initial tumor uptake of ¹¹¹In(DOTA-2P4G-RGD₄) (11.4 %ID/g at 0.5 h p.i.) was comparable to that of ¹¹¹In(DOTA-6P-RGD₄) (10.5 %ID/g at 0.5 h p.i.) within the experimental error; but its tumor radioactivity washout was slightly faster with t_1/2 being ~35 h. ¹¹¹In(DOTA-2P4G-RGD₄) also showed high tumor/muscle ratio (Table 5), and a rapid liver clearance (3.6 and 0.25 %ID/g at 0.5 and 72 h p.i., respectively). As a result, ¹¹¹In(DOTA-2P4G-RGD₄) has the highest tumor/liver ratio at 72 h p.i. (Figure 3). ¹¹¹In(DOTA-2P4G-RGD₄) also had very high initial intestine uptake; but it was much lower (p <0.01) than that of ¹¹¹In(DOTA-6P-RGD₄) at >1 h p.i. The tumor uptake of ¹¹¹In(DOTA-2P-RGD₄) was also high and close to that of ¹¹¹In(DOTA-6P-RGD₄) over the 72 h period (Figure 3). Its intestine uptake was comparable to that of ¹¹¹In(DOTA-6P-RGD₄) within the experimental errors (Figure 3). ¹¹¹In(DOTA-2P-RGD₄) had a rapid clearance from normal organs, such as the blood, kidneys and liver. The tumor/liver and tumor/kidney ratios of ¹¹¹In(DOTA-2P-RGD₄) were well comparable to those obtained for ¹¹¹In(DOTA-6P-RGD₄) and ¹¹¹In(DOTA-6G-RGD₄) (Figure 3).

It seems that ¹¹¹In(DOTA-2P-RGD₄), ¹¹¹In(DOTA-2P4G-RGD₄) and ¹¹¹In(DOTA-6P-RGD₄) might have higher tumor uptake than ¹¹¹In(DOTA-3P-RGD₂) over the first 24 h, but their tumor uptake differences were not significant within the experimental error (p > 0.05). The 72 h tumor uptake follows the general order of ¹¹¹In(DOTA-6P-RGD₄) (3.04 %ID/g) > ¹¹¹In(DOTA-2P-RGD₄) (2.87 %ID/g) ~ ¹¹¹In(DOTA-2P4G-RGD₄) (2.66 %ID/g) > ¹¹¹In(DOTA-3P-RGD₂) (2.18 %ID/g), which is similar to the trend of integrin α_vβ₃ binding affinity of DOTA-6P-RGD₄ (IC₅₀ = 0.3 ± 0.1 nM) ~ DOTA-2P4G-RGD₄ (IC₅₀ = 0.2 ± 0.1 nM) ~ DOTA-2P-RGD₄ (IC₅₀ = 0.5 ± 0.1 nM) > DOTA-3P-RGD₂ (IC₅₀ = 1.5 ± 0.2 nM). Among the ¹¹¹In radiotracers evaluated in this animal model, ¹¹¹In(DOTA-6G-RGD₄) had the highest uptake in the tumor and intestine (Figure 3) at 72 h p.i. The half-life of ¹¹¹In(DOTA-6G-RGD₄) in the tumor was estimated to be 60 h. In contrast, ¹¹¹In(DOTA-3P-RGD₂) and ¹¹¹In(DOTA-2P4G-RGD₄) had low uptake in the intestine, kidneys and liver over the 72 h period. This was particularly true at > 24 h p.i. As a result, ¹¹¹In(DOTA-3P-RGD₂) and ¹¹¹In(DOTA-2P4G-RGD₄) had the tumor/kidney and tumor/liver ratios that were significantly better (p < 0.05) than those of ¹¹¹In(DOTA-6G-RGD₄) and ¹¹¹In(DOTA-6P-RGD₄) during that period of time (Figure 3).

As expected, ¹¹¹In(DOTA-P-RGD) had the lowest tumor uptake (3.7 and 0.7 %ID/g at 0.5 and 72 h p.i., respectively) among ¹¹¹In radiotracers evaluated in this study. ¹¹¹In(DOTA-P-RGD₂) had a relatively high tumor uptake (6.1 at 0.5 h p.i.), but it had a significant washout from the tumor with uptake values being 4.9, 4.8, 3.1 and 1.6 %ID/g at 1, 4, 24 and 72 h p.i., respectively. The tumor uptake follows the trend of ¹¹¹In(DOTA-3P-RGD₂) > ¹¹¹In(DOTA-P-RGD₂) > ¹¹¹In(DOTA-P-RGD), which is completely consistent with the order of their α_vβ₃ binding affinity (IC₅₀ value: DOTA-3P-RGD₂ < DOTA-P-RGD₂ < DOTA-P-RGD).

Integrin α_vβ₃ Specificity. Figure 4A compares the 60-min organ uptake of ¹¹¹In(DOTA-6P-RGD₄) in the absence/presence of excess RGD₂ (~350 μg/mouse). Co-injection of excess RGD₂ significantly blocked the tumor uptake of ¹¹¹In(DOTA-6P-RGD₄) (0.7 %ID/g with RGD₂ vs. 11.1 %ID/g without RGD₂ at 1 h p.i.). The normal organ uptake of ¹¹¹In(DOTA-6P-RGD₄) was also blocked by the presence of excess RGD₂. For example, the uptake of ¹¹¹In(DOTA-6P-RGD₄) in the eyes, heart, intestine, liver, lungs and spleen was 1.4, 0.8, 11.5, 4.0, 3.7 and 2.1 %ID/g, respectively, without excess RGD₂, while its uptake values in the same organs were 0.1, 0.4, 0.5, 0.7, 1.2, and 0.5 %ID/g, respectively, with excess RGD₂. Similar properties were also observed for other radiolabeled (^99mTc, ⁶⁴Cu and ¹¹¹In) cyclic RGD dimers [18, 25, 36-38, 43, 45] and tetramers [20, 31, 46].

 Figure 4 

A: Comparison of the 60-min biodistribution data of ¹¹¹In(DOTA-6P-RGD₄) in the athymic nude mice (n = 5) bearing U87MG human glioma xenografts in the absence/presence of excess RGD₂ to demonstrate its integrin α_vβ₃-specificity. B: Comparison of the 60-min biodistribution data of ¹¹¹In(DOTA-6P-RGD₄) and ¹¹¹In(DOTA-6P-RGK₄) in the athymic nude mice (n = 5) bearing U87MG glioma xenografts to demonstrate the RGD-specificity.

RGD Specificity. Figure 4B compares the 60-min uptake of ¹¹¹In(DOTA-6P-RGD₄) and ¹¹¹In(DOTA-6P-RGK₄) in the U87MG glioma and normal organs. As expected, ¹¹¹In(DOTA-6P-RGK₄) had much lower (p < 0.01) tumor uptake (0.8 %ID/g) than ¹¹¹In(DOTA-6P-RGD₄) (11.1 %ID/g) due to the low integrin α_vβ₃ binding affinity of DOTA-6P-RGK₄. ¹¹¹In(DOTA-6P-RGK₄) also had significantly lower uptake than ¹¹¹In(DOTA-6P-RGD₄) in the normal organs, such as intestine, kidneys, liver, lungs and spleen (Figure 4B). Apparently, replacing c(RGDfK) motifs with c(RGKfD) moieties resulting in a dramatic uptake decrease in the tumor and integrin α_vβ₃-positive organs, particularly intestine, liver, lungs and spleen.

Integrin α_vβ₃ Expression in Normal Organs. To further understand the localization mechanism of ¹¹¹In radiotracers, the tumor, lung, liver and kidney tissues were harvested, and were then double-stained for the CD31 and integrin β₃ expression. As shown in Figure 5A, U87MG glioma had a high integrin β₃ expression level on both tumor cells and tumor neovasculature. Most of the blood vessels expressing integrin β₃ were seen from the overlay of positive CD31 and β₃ positive staining. Both the liver and lungs had much lower integrin β₃ expression levels than the U87MG tumor. The integrin β₃ expression in kidneys was very low, which seems consistent with the results from blocking experiment (Figure 4A). These results were further confirmed by the Western blot data (Figure 5B).

Imaging Study. Figure 6 illustrates whole-body images of the glioma-bearing mice administered with ¹¹¹In(DOTA-2P-RGD₄), ¹¹¹In(DOTA-2P4G-RGD₄) and ¹¹¹In(DOTA-6P-RGD₄ at 1, 4, 24 and 72 h p.i. The glioma tumors could be clearly visualized with excellent T/B contrast as early as 1 h after injection of ¹¹¹In(DOTA-2P-RGD₄), ¹¹¹In(DOTA-2P4G-RGD₄) and ¹¹¹In(DOTA-6P-RGD₄). All three radiotracers had a long tumor retention time with t_1/2 > 30 h, which was completely consistent with the results obtained from ex-vivo biodistribution studies (Tables 2-4).

Metabolic Stability. Figure 7 shows typical radio-HPLC chromatograms of ¹¹¹In(DOTA-2P-RGD₄) and ¹¹¹In(DOTA-6P-RGD₄) in saline before injection, in urine at 30 min and 120 min p.i., and in feces at 120 min p.i. The radioactivity recovery from the urine and feces samples was >95% in both cases. Attempts to extract radioactivity from the liver and kidneys were not successful due to very limited radioactivity accumulation in both organs. There was little metabolism detected in the urine or feces samples over the 2 h study period for ¹¹¹In(DOTA-2P-RGD₄). ¹¹¹In(DOTA-2P4G-RGD₄) had the same metabolic stability as ¹¹¹In(DOTA-2P-RGD₄). ¹¹¹In(DOTA-6P-RGD₄) was able to maintain its integrity during its excretion via the renal route. However, there was a significant metabolism during its excretion via the hepatobiliary route. As a matter of fact, only ~45% of ¹¹¹In(DOTA-6P-RGD₄) remained intact in the feces sample. The metabolic instability was also reported for ¹¹¹In(DOTA-6G-RGD₄) [46].

 Figure 5 

A: Representative microscopic fluorescence images of the organ tissues (U87MG glioma, lung, liver and kidney) obtained from the U87MG tumor-bearing mice. For β3 staining (red), frozen tissue slices were incubated with a β3 primary antibody followed by a DyLight 594-conjugated secondary antibody. For CD31 staining (green), slices were incubated with a CD31 primary antibody followed by a FITC-conjugated secondary antibody (×100). Yellow color indicates the overlay of integrin β3 and CD31. Phase contrast pictures were shown as histological reference. B: Western blot results showing expression of integrin β₃ in U87MG glioma, lungs, liver and kidneys obtained from the U87MG tumor-bearing mice.

 Figure 6 

The whole-body planar images of the tumor-bearing mice (bearing U87MG human glioma xenografts) administered with ~100 μCi of ¹¹¹In(DOTA-3P-RGD₂) (top), ¹¹¹In(DOTA-2P-RGD₄) (middle) and ¹¹¹In(DOTA-6P-RGD₄) (bottom) to illustrate the long tumor retention times of ¹¹¹In-labeled cyclic RGD tetramers. Arrows indicate the presence of tumors.

 Figure 7 

Representative radio-HPLC chromatograms of ¹¹¹In(DOTA-2P-RGD₄) (left) and ¹¹¹In(DOTA-6P-RGD₄) (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. ¹¹¹In(DOTA-2P4G-RGD₄) has the same metabolic stability in the same animal model.

Discussion

In this study, we successfully prepared ¹¹¹In complexes of DOTA-P-RGD, DOTA-P-RGD₂, DOTA-2P-RGD₄, DOTA-2P4G-RGD₄ and DOTA-6P-RGD₄. We found that their integrin α_vβ₃ binding affinity follows the order: DOTA-6P-RGD₄ ~ DOTA-2P4G-RGD₄ ~ DOTA-2P-RGD₄ > DOTA-P-RGD₂ > DOTA-P-RGD (Figure 2). Previously, we showed that DOTA-3P-RGD₂ binds to the integrin α_vβ₃ on U87MG glioma cells in a bivalent fashion due to the increased distance (38 bonds excluding lysine residues) between two cyclic RGD motifs [38, 45]. This study further confirms this observation. For example, the integrin α_vβ₃ binding affinity of DOTA-3P-RGD₂ (IC₅₀ = 1.5 ± 0.2 nM) is significantly higher (p < 0.01) than that of both DOTA-P-RGD (IC₅₀ = 44.3 ± 3.5 nM) and DOTA-P-RGD₂ (IC₅₀ = 5.0 ± 1.0 nM). The bivalency is further supported by the higher tumor uptake of ¹¹¹In(DOTA-3P-RGD₂) than ¹¹¹In(DOTA-P-RGD₂) and ¹¹¹In(DOTA-P-RGD) over the 72 h study period (Figure 3).

The integrin α_vβ₃ binding affinities of DOTA-2P-RGD₄, DOTA-2P4G-RGD₄ and DOTA-6P-RGD₄ are only marginally higher than that of DOTA-3P-RGD₂ (Figure 2), suggesting that they might share the same bivalency in binding to the integrin α_vβ₃. This conclusion is completely consistent with the similar tumor uptake of ¹¹¹In(DOTA-3P-RGD₂), ¹¹¹In(DOTA-2P-RGD₄), ¹¹¹In(DOTA-2P4G-RGD₄) and ¹¹¹In(DOTA-6P-RGD₄) over the first 24 h (Figure 3). If DOTA-2P-RGD₄, DOTA-2P4G-RGD₄ and DOTA-6P-RGD₄ were tetravalent, they would have had much better integrin α_vβ₃ binding affinity than DOTA-3P-RGD₂ while ¹¹¹In(DOTA-2P-RGD₄), ¹¹¹In(DOTA-2P4G-RGD₄) and ¹¹¹In(DOTA-6P-RGD₄) would have had significantly higher initial tumor uptake than that of ¹¹¹In(DOTA-3P-RGD₂).

Two factors (bivalency and the “locally enriched” RGD concentration) contribute to the higher α_vβ₃ binding affinity of multimeric RGD peptides than that of their monomeric counterpart, and the higher tumor uptake of radiolabeled RGD dimers and tetramers than their monomeric analogs [15, 40-46]. The concentration factor exists in all dimers and tetramers. Because of the short distance between two RGD motifs in RGD₂, it is unlikely for DOTA-P-RGD₂ to be bivalent [15]. However, once the first one is bonded, the “local concentration” of second RGD motif will be dramatically increased in the vicinity of neighboring integrin α_vβ₃ sites. This may explain why DOTA-P-RGD₂ has the better integrin α_vβ₃ binding affinity (Figure 2) than DOTA-P-RGD, and ¹¹¹In(DOTA-P-RGD₂) has higher tumor uptake (Figure 3) than ¹¹¹In(DOTA-P-RGD). Since IC₅₀ values follow the order of DOTA-3P-RGD₂ > DOTA-P-RGD₂ > DOTA-P-RGD (Figure 2), we believe that both bivalency and concentration factors contribute to the higher tumor uptake of ¹¹¹In(DOTA-3P-RGD₂) than ¹¹¹In(DOTA-P-RGD) (Figure 3).

DOTA-3P-RGD₂ has two cyclic RGD motifs with only one possibility to achieve the bivalency. In DOTA-2P-RGD₄, DOTA-2P4G-RGD₄ and DOTA-6P-RGD₄, however, any two of the four RGD motifs can achieve bivalency due to the longer distance between them. Even though DOTA-2P-RGD₄, DOTA-2P4G-RGD₄ and DOTA-6P-RGD₄ may not be tetravalent, the two extra RGD motifs might contribute to their better integrin α_vβ₃ binding affinity than that of DOTA-3P-RGD₂ (Figure 2), and the slightly higher 72-h tumor uptake (Figure 3) for ¹¹¹In(DOTA-6P-RGD₄) than that for ¹¹¹In(DOTA-3P-RGD₂). Higher tumor uptake was also observed for ¹¹¹In(DOTA-6G-RGD₄) than that of ¹¹¹In(DOTA-3P-RGD₃) at 72 h p.i. in the same tumor-bearing animal model [46].

It is very important to note that the ability of a multimeric cyclic RGD peptide to achieve bivalency depends largely on the integrin α_vβ₃ density on tumor cells and tumor neovasculature. If the integrin α_vβ₃ density is high, the distance between two neighboring integrin α_vβ₃ sites will be short, which makes it easier for a multimeric RGD peptide to achieve bivalency. If the integrin α_vβ₃ density is low, the distance between two neighboring integrin α_vβ₃ sites will be long, and it might be more difficult for the same multimeric RGD peptide to achieve simultaneous integrin α_vβ₃ binding. In that situation, the “concentration factor” will be the dominant force contributing to the better integrin α_vβ₃ binding affinity of a multimeric cyclic RGD peptide than that of its monomeric counterpart, and to the higher tumor uptake of the radiolabeled cyclic RGD multimers as compared to their monomeric analogs.

The integrin α_vβ₃-specificity of ¹¹¹In(DOTA-6P-RGD₄) was demonstrated by using RGD₂ as the blocking agent. The uptake blockage of ¹¹¹In(DOTA-6P-RGD₄) in the tumor (Figure 4A) suggests that its glioma uptake is indeed mainly integrin α_vβ₃-mediated. The partial blockage of the uptake for ¹¹¹In(DOTA-6P-RGD₄) in eyes, intestine, kidneys, lungs, liver and spleen indicates that the accumulation of ¹¹¹In(DOTA-6P-RGD₄) in these organs is partially integrin α_vβ₃-mediated. This conclusion is supported by the tissue-staining studies (Figure 5A). The low integrin β₃ expression in kidneys (Figure 5B) suggests that the radiotracer kidney uptake is caused mainly by tubular re-absorption instead of specific integrin α_vβ₃ binding. The RGD-specificity of ¹¹¹In(DOTA-6P-RGD₄) was demonstrated by the extremely low integrin α_vβ₃ binding affinity of DOTA-6P-RGK₄ (Figure 2: IC₅₀ = 437 ± 35 nM), and the significantly lower tumor uptake (Figure 4B) of ¹¹¹In(DOTA-6P-RGK₄) as compared to that of ¹¹¹In(DOTA-6P-RGD₄). In addition, ¹¹¹In(DOTA-6P-RGK₄) also has low uptake in the intestine, kidneys, liver, lungs and spleen, suggesting that the high uptake of ¹¹¹In(DOTA-6P-RGD₄) in these organs is also RGD-specific.

Non-invasive imaging of integrin α_vβ₃ is highly desirable for patient selection before anti-angiogenic treatment and for more effective monitoring of therapeutic efficacy in the integrin α_vβ₃-positive cancer patients [47-49]. For the tumors to be detectable by SPECT (single photon emission tomography) or PET (positron emission tomography), they must have sufficient accumulation of radioactivity. In this study, we found that small tumors (0.03 - 0.05 g) were clearly visualized (Figure 6) with good contrast in the tumor-bearing mice administered with ¹¹¹In(DOTA-2P-RGD₄), ¹¹¹In(DOTA-2P4G-RGD₄) and ¹¹¹In(DOTA-6P-RGD₄). The tumor detection limit was 3 - 4 mm in diameter using a modified clinical γ-camera. We believe that the tumor detection limit might be < 3 mm using the higher resolution SPECT cameras or SPECT/CT systems with ¹¹¹In(DOTA-3P-RGD₂), ¹¹¹In(DOTA-2P-RGD₄), ¹¹¹In(DOTA-2P4G-RGD₄) and ¹¹¹In(DOTA-6P-RGD₄) as radiotracers. From this view point, all these ¹¹¹In radiotracers are useful for the early detection of integrin α_vβ₃-positive tumors.

It is well-established that multimerization of cyclic RGD peptides can significantly improve their integrin α_vβ₃-targeting capability [9, 15-41]. However this often leads to more accumulation of the radiolabeled RGD multimers in the integrin α_vβ₃-positive organs, as evidenced by the higher uptake of ¹¹¹In(DOTA-2P-RGD₄) and ¹¹¹In(DOTA-6P-RGD₄) than ¹¹¹In(DOTA-3P-RGD₂) in the intestine, liver and kidneys. As a result, ¹¹¹In(DOTA-2P-RGD₄) and ¹¹¹In(DOTA-6P-RGD₄) have the tumor/liver and tumor/kidney ratios significantly (p < 0.01) lower than those of ¹¹¹In(DOTA-3P-RGD₂) (Figure 3). On the basis of tumor-to-background ratios, we believe that ¹¹¹In(DOTA-3P-RGD₂) is a better diagnostic radiotracer than ¹¹¹In(DOTA-2P-RGD₄) and ¹¹¹In(DOTA-6P-RGD₄). At this moment, we are not clear why ¹¹¹In(DOTA-2P4G-RGD₄) has lower (p < 0.05) intestine uptake than ¹¹¹In(DOTA-6P-RGD₄) and ¹¹¹In(DOTA-6G-RGD₄) (Figure 3) even though they share the same four cyclic RGD motifs. However, it is clear that the G₃ and PEG₄ linkers between cyclic RGD motifs have a significant impact on biodistribution characteristics of ¹¹¹In-labeled cyclic RGD tetramers.

For diagnostic radiotracers, the contrast between the targeted organ and surrounding tissues is the first priority. For therapeutic radiotracers, however, maximal delivery of tumoricidal radiation dose becomes critically important. The radiotracer with longer retention time will deliver higher radiation dose to tumors during the same period of time. From this viewpoint, DOTA-6G-RGD₄ remains the best for the integrin α_vβ₃-targeted therapeutic ⁹⁰Y and ¹⁷⁷Lu radiotracers since ¹¹¹In(DOTA-6G-RGD₄) has the longest retention time (t_1/2 ~ 60 h) among four ¹¹¹In-labeled RGD tetramers (Figure 3). However, one cannot totally ignore its high intestine uptake and low tumor/liver ratios over the 72 h period. Considering the radiotracer uptake in normal organs (particularly intestine and liver), we believe that DOTA-3P-RGD₂ and DOTA-2P4G-RGD₄ have significant advantages over DOTA-6P-RGD₄ and DOTA-6G-RGD₄ because the intestine and liver uptake of ¹¹¹In(DOTA-3P-RGD₂) and ¹¹¹In(DOTA-2P4G-RGD₄) is significantly lower than that of ¹¹¹In(DOTA-6P-RGD₄) and ¹¹¹In(DOTA-6P-RGD₄) (Figure 3). It must be noted that there is always a subtle balance between maximizing the radiation dose to tumor cells and minimizing the radiation burden to the surrounding normal tissues or organs.

It is interesting to point out that ¹¹¹In(DOTA-2P-RGD₄) is stable during its renal and hepatobiliary excretion over the 2 h study period (Figure 7). In contrast, ¹¹¹In(DOTA-6P-RGD₄) has a significant metabolism during its hepatobiliary excretion while it remains relatively unchanged during its renal excretion (Figure 7). ¹¹¹In(DOTA-6G-RGD₄) has similar metabolic instability except that only 25% of it remains intact in the feces samples in the same animal model [46]. Although it remains unclear as for what causes this metabolic instability, one thing is clear that the linkers between RGD motifs have a significant impact on the metabolic stability of ¹¹¹In-labeled cyclic RGD tetramers.

Conclusion

In summary, we evaluated five new radiotracers ¹¹¹In(L) (L = DOTA-P-RGD, DOTA-P-RGD₂, DOTA-2P-RGD₄, DOTA-2P4G-RGD₄ and DOTA-6P-RGD₄) to explore the effect of peptide and PEG₄ linker multiplicity on their tumor uptake and T/B ratios. We found that the addition of two PEG₄ linkers between two RGD motifs significantly improves the integrin α_vβ₃-targeting capability of cyclic RGD dimers as evidenced by their higher integrin α_vβ₃-binding affinity and the high tumor uptake and better T/B ratios of their radiotracers. We also found that DOTA-2P-RGD₄, DOTA-2P4G-RGD₄ and DOTA-6P-RGD₄ are not tetravalent in binding to integrin α_vβ₃ because increasing the RGD multiplicity from dimers to tetramers didn't significantly improve the radiotracer's initial tumor uptake. However, the extra RGD motifs in DOTA-2P-RGD₄, DOTA-2P4G-RGD₄ and DOTA-6P-RGD₄ might contribute to slightly higher tumor uptake of ¹¹¹In(DOTA-2P-RGD₄), ¹¹¹In(DOTA-2P4G-RGD₄) and ¹¹¹In(DOTA-6P-RGD₄) than that of ¹¹¹In(DOTA-3P-RGD₂) at 72 h p.i. Among the ¹¹¹In-labeled cyclic RGD tetramers evaluated in this animal model, ¹¹¹In(DOTA-2P4G-RGD₄) has the best tumor/kidney and tumor/liver ratios. The combination of its high tumor uptake, long retention time and low uptake in normal organs suggests that ⁹⁰Y(DOTA-2P4G-RGD₄) and ¹⁷⁷Lu(DOTA-2P4G-RGD₄) may have the potential for radiotherapy of integrin α_vβ₃-positive tumors [50]. Evaluations of their therapeutic efficacy are still in progress, and the result from these studies will be communicated elsewhere.

Abbreviations

c(RGDfK): cyclo(Lys-Arg-Gly-Asp-D-Phe); c(RGDyK): cyclo(Lys-Arg-Gly-Asp-D-Tyr);

E[c(RGDfK)]₂ (RGD₂): Glu[cyclo(Lys-Arg-Gly-Asp-D-Phe)-cyclo(Lys-Arg-Gly-Asp-D-Phe)];

DOTA: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid;

PEG₄: 15 amino-4,710,13-tetraoxapentadecanoic acid;

PEG₄-E[G₃-c(RGDfK)]₂: PEG₄-Glu{cyclo[Lys(Gly-Gly-Gly)-Arg-Gly-Asp-D-Phe]}-cyclo[Lys(Gly-Gly-Gly)-Arg-Gly-Asp-D-Phe]};

PEG₄-E[PEG₄-c(RGDfK)]₂: PEG₄-Glu{cyclo[Lys(PEG₄)-Arg-Gly-Asp-D-Phe]-cyclo[Lys(PEG₄)-Arg-Gly-Asp-D-Phe]};

PEG₄-E[PEG₄-c(RGKfD)]₂: PEG₄-Glu{cyclo[Arg-Gly-Lys(PEG₄)-D-Phe-Asp]-cyclo[Arg-Gly-Lys(PEG₄)-D-Phe-Asp]};

E{E[c(RGDfK)]₂}₂ (RGD₄): Glu{Glu[cyclo(Lys-Arg-Gly-Asp-D-Phe)]-cyclo(Lys-Arg-Gly-Asp-D-Phe)}-{Glu[cyclo(Lys-Arg-Gly-Asp-D-Phe)]-cyclo(Lys-Arg-Gly-Asp-D-Phe)};

E{PEG₄-E[c(RGDfK)]₂}₂: Glu{PEG₄-Glu[cyclo(Lys-Arg-Gly-Asp-D-Phe)]-cyclo(Lys-Arg-Gly-Asp-D-Phe)}-{PEG₄-Glu[cyclo(Lys-Arg-Gly-Asp-D-Phe)]-cyclo(Lys-Arg-Gly-Asp-D-Phe)};

E{PEG₄-E[G₃-c(RGDfK)]₂}₂: Glu{PEG₄-Glu[cyclo(Lys(Gly-Gly-Gly)-Arg-Gly-Asp-D-Phe)]-cyclo(Lys(Gly-Gly-Gly)-Arg-Gly-Asp-D-Phe)}-{PEG₄-Glu[cyclo(Lys(Gly-Gly-Gly)-Arg-Gly-Asp-D-Phe)]-cyclo(Lys(Gly-Gly-Gly)-Arg-Gly-Asp-D-Phe)};

E{PEG₄-E[PEG₄-c(RGDfK)]₂}₂: Glu{PEG₄-Glu[cyclo(Lys(PEG₄)-Arg-Gly-Asp-D-Phe)]-cyclo(Lys(PEG₄)-Arg-Gly-Asp-D-Phe)}-{PEG₄-Glu[cyclo(Lys(PEG₄)-Arg-Gly-Asp-D-Phe)]-cyclo(Lys(PEG₄)-Arg-Gly-Asp-D-Phe)};

E{PEG₄-E[PEG₄-c(RGKfD)]₂}₂: Glu{PEG₄-Glu[cyclo(Arg-Gly-Lys(PEG₄)-D-Phe-Asp)]-cyclo(Arg-Gly-Lys(PEG₄)-D-Phe-Asp)}-{PEG₄-Glu[cyclo(Arg-Gly-Lys(PEG₄)-D-Phe-Asp)]-cyclo(Arg-Gly-Lys(PEG₄)-D-Phe-Asp)}.

Acknowledgements

This work is supported, in part, by Purdue University and the research grant: R01 CA115883 from the National Cancer Institute (NCI).

Conflict of Interest

The authors have declared that no conflict of interest exists.

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Author contact

Corresponding author: School of Health Sciences, Purdue University, 550 Stadium Mall Drive, West Lafayette, IN 47907. Phone: 765-494-0236; Fax 765-496-1377; Email: liu100edu

Received 2011-6-30
Accepted 2011-7-18
Published 2011-7-25

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