Evaluation of a 99mTc-Labeled Cyclic RGD Tetramer for Noninvasive

Mar 7, 2007 - SPECT images displayed significant radiotracer localization in tumor with good contrast as early as 1 h p.i. The high tumor uptake and f...
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Bioconjugate Chem. 2007, 18, 438−446

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Evaluation of a 99mTc-Labeled Cyclic RGD Tetramer for Noninvasive Imaging Integrin rvβ3-Positive Breast Cancer Shuang Liu,*,† Wen-Yuan Hsieh,† Young Jiang,† Young-Seung Kim,† Subramanya G. Sreerama,† Xiaoyuan Chen,‡ Bing Jia,§ and Fan Wang§ School of Health Sciences, Purdue University, West Lafayette, Indiana 47907-2051, Molecular Imaging Program at Stanford, Department of Radiology & Bio-X, Stanford University, Stanford, California 94305-5344, Medical Isotopes Research Center, Peking University, Beijing 100083, China. Received October 4, 2006; Revised Manuscript Received January 10, 2007

Integrin Rvβ3 plays a critical role in tumor angiogenesis and metastasis. Radiolabeled RGD peptides that are integrin Rvβ3-specific are very useful for noninvasive imaging of integrin expression in rapidly growing and metastatic tumors. In this study, we determined the binding affinity of E{E[c(RGDfK)]2}2 (tetramer) and its 6-hydrazinonicotinamide conjugate (HYNIC-tetramer) against the binding of 125I-echistatin to the integrin Rvβ3positive MDA-MB-435 breast cancer cells. The athymic nude mice bearing MDA-MB-435 xenografts were used to evaluate the potential of ternary ligand complex [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] (TPPTS ) trisodium triphenylphosphine-3,3′,3′′-trisulfonate) as a new radiotracer for imaging breast cancer integrin Rvβ3 expression by single photon emission computed tomography (SPECT). It was found that the binding affinity of tetramer (IC50 ) 51 ( 11 nM) was slightly higher than that of its dimeric analogue (IC50 ) 78 ( 27 nM) and is comparable to that of the HYNIC-tetramer conjugate (IC50 ) 55 ( 11 nM) within the experimental error. Biodistribution data showed that [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] had a rapid blood clearance (4.61 ( 0.81 %ID/g at 5 min postinjection (p.i.) and 0.56 ( 0.12 %ID/g at 120 min p.i.) and was excreted mainly via the renal route. [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] had high tumor uptake with a long tumor retention (5.60 ( 0.87 %ID/g and 7.30 ( 1.32 %ID/g at 5 and 120 min p.i., respectively). The integrin Rvβ3-specificity was demonstrated by co-injection of excess E[c(RGDfK)]2, which resulted in a significant reduction in tumor uptake of the radiotracer. The metabolic stability of [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] was determined by analyzing urine and feces samples from the tumor-bearing mice at 120 min p.i. In the urine, about 20% of [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] remained intact while only ∼15% metabolized species was detected in feces. SPECT images displayed significant radiotracer localization in tumor with good contrast as early as 1 h p.i. The high tumor uptake and fast renal excretion make [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] a promising radiotracer for noninvasive imaging of the integrin Rvβ3-positive tumors by SPECT.

INTRODUCTION Angiogenesis is a requirement for tumor growth and metastasis (1-5). Without the neovasculature to provide oxygen and nutrients, tumors cannot grow beyond 1-2 mm in size. Once vascularized, previously dormant tumors begin to grow rapidly and their volumes increase exponentially. Angiogenic process depends on vascular endothelial cell migration and invasion and is regulated by cell adhesion receptors. Integrins are such a family of proteins that facilitate cellular adhesion to and migration on extracellular matrix proteins found in intercellular spaces and basement membranes and regulate cellular entry and withdrawl from the cell cycle (4-7). Integrin Rvβ3 is a receptor for the extracellular matrix proteins with an exposed arginine-glycine-aspartic (RGD) tripeptide sequence (5, 6). Integrin Rvβ3 is normally expressed at low levels on epithelial cells and mature endothelial cells but it is highly expressed on the activated endothelial cells in the neovasculature of tumors, including osteosarcomas, glioblastomas, melanomas, lung carcinomas, and breast cancer (8-14). It has been demonstrated * To whom correspondence should be addressed. School of Health Sciences, Purdue University, 550 Stadium Mall Dr., West Lafayette, IN 47907. Phone: 765-494-0236; Fax: 765-496-1377; E-mail: lius@ pharmacy.purdue.edu. † Purdue University. ‡ Stanford University. § Peking University.

that integrin Rvβ3 is overexpressed on both endothelial and tumor cells in human breast cancer xenografts (14). The integrin Rvβ3 expression correlates well with tumor progression and invasiveness of melanoma, glioma, and breast cancers (8, 9, 13). The highly restricted expression of integrin Rvβ3 during tumor growth, invasion, and metastasis present an interesting molecular target for early detection of rapidly growing and metastatic tumors (15-22). We and others have been using multimeric cyclic RGD peptides to develop the integrin Rvβ3-targeted radiotracers to image rapidly growing and metastatic tumors (23-32). The RGD peptides serve as targeting biomolecules to carry radionuclide (e.g., 99mTc and 64Cu) to the integrin Rvβ3 overexpressed on tumor cells and endothelial cells of tumor neovasculature. Recently, we reported 64Cu-DOTA-E{E[c(RGDfK)]2}2 as a positron emission tomography (PET) radiotracer to image glioma integrin Rvβ3 expression (26). Results from the in vitro assay showed that the tetramer, E{E[c(RGDfK)]2}2, had a higher integrin Rvβ3 binding affinity than the dimer, E[c(RGDfK)]2, against 125I-echistatin due to increased peptide valency. As a result, 64Cu-DOTA-E{E[c(RGDfK)]2}2 showed very high tumor uptake and a long tumor retention (9.93 ( 1.05 %ID/g at 30 min and 4.56 ( 0.51 %ID/g at 24 h p.i.). To further illustrate the advantage of the tetramer, three DOTA conjugates (DOTA-E-c(RGDfK), DOTA-E[c(RGDfK)]2, DOTA-E{E[c(RGDfK)]2}2) and their 111In complexes were prepared (32). Biodistribution studies on

10.1021/bc0603081 CCC: $37.00 © 2007 American Chemical Society Published on Web 03/07/2007

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Figure 1. Schematic structure of [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] (tetramer ) E{E[c(RGDfK)]2}2; TPPTS ) trisodium triphenylphosphine3,3′,3′′-trisulfonate).

111In-DOTA-E-c(RGDfK), 111In-DOTA-E-[c(RGDfK)] , and 2 111In-DOTA-E{E[c(RGDfK)] } were performed in athymic 2 2

nude mice bearing subcutaneous SK-RC-52 tumors. At 8 h p.i, 2}2 showed a much higher tumor uptake (7.40 ( 1.12 %ID/g) than the monomer (2.30 ( 0.34 %ID/g) and the dimer (5.17 ( 1.22 %ID/g). It was concluded that the tetramer E{E[c(RGDfK)]2}2 is the best targeting biomolecule with respect to radiotracer tumor uptake and tumorto-background (T/B) ratios (32). These promising results led us to prepare the 6-hydrazinonicotinamide-conjugated tetramer (HYNIC-tetramer) and its 99mTc complex [99mTc(HYNICtetramer)(tricine)(TPPTS)] (Figure 1: TPPTS ) trisodium triphenylphosphine-3,3′,3′′-trisulfonate). 99mTc is the ideal radionuclide for SPECT imaging due to its optimal nuclear properties (t1/2 ) 6.02 h and Emax ) 142 KeV) and easy availability at low cost. HYNIC is used as bifunctional coupling agent for the 99mTc-labeling. TPPTS is used as the coligand because of its three negative charges and the high solution stability of its 99mTc complexes (33). We now report the evaluation of [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] as a SPECT radiotracer to image the integrin Rvβ3 expression in athymic nude mice bearing MDA-MB-435 human breast cancer xenografts. The objective is to explore the impact of peptide multiplicity on biodistribution characteristics and metabolism of the 99mTc-labeled multimeric cyclic RGDfK peptides. The results from this study will allow us to further demonstrate the superiority of the cyclic RGDfK tetramer over its dimeric and monomeric analogues with respect to tumortargeting capability and in vivo kinetics. 111In-DOTA-E{E[c(RGDfK)]

MATERIALS AND METHODS Trisodium triphenylphosphine-3,3′,3′′-trisulfonate (TPPTS) and tricine were purchased from Sigma-Aldrich. E{E[c(RGDfK)]2}2 was prepared using the reported procedure (26). Sodium succinimidyl 6-(2-(2-sulfonatobenzaldehyde)hydrazono)nicotinate (HYNIC-NHS) was prepared according to literature method (34). Na99mTcO4 was obtained from a commercial DuPont Pharma 99Mo/99mTc generator, N. Billerica, MA. Mass spectral data were collected on a Finnigan LCQ classic mass spectrometer, School of Pharmacy, Purdue University. HPLC Method 1 used a LabAlliance semiprep HPLC system equipped with a UV-vis detector (λ ) 254 nm) and Zorbax C18 semiprep column (9.4 mm × 250 mm, 100 Å pore size). The flow rate was 2.5 mL/min. The mobile phase was isocratic with 90% solvent A (0.1% acetic acid in water) and 10% solvent B (0.1% acetic acid in acetonitrile) at 0-5 min, followed by a gradient mobile phase going from 90% solvent A and 10% solvent B at 5 min to 60% solvent A and 40% solvent B at 20 min. The radio-HPLC method (Method 2) used the LabAlliance HPLC system equipped with a β-ram IN-US detector and Zorbax C18 column (4.6 mm × 250 mm, 300 Å pore size). The flow rate was 1 mL/min. The mobile phase was isocratic with 90% solvent A (25 mM ammonium acetate buffer, pH ) 5.0) and 10% solvent B (acetonitrile) at 0-2 min, followed by a gradient mobile phase going from 10% solvent B at 2 min to 15% solvent B at 5 min and to 20% solvent B at 20 min. Synthesis of HYNIC-E{E[c(RGDfK)]2}2 (HYNIC-tetramer). HYNIC-NHS (4.17 mg, 10 µmol) and E{E[c(RGDfK)]2}2

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(14.0 mg, 10.6 µmol) were dissolved in a mixture of DMF and water (50:50 ) v:v). The pH was adjusted to 8.5-9.0 with 0.1 N NaOH. The mixture was incubated overnight at room temperature. The product was purified by HPLC (Method 1). The peak of interest at ∼14 min was collected. The collected fractions were combined, and lyophilized to give a pale yellow powder. The yield was 11 mg (∼65%) with the purity >95% by HPLC. ESI-MS: C136H188N42O38S, calculated 3051.30, observed 3050.93 ([M + H]+). Synthesis of [99mTc(HYNIC-tetramer)(tricine)(TPPTS)]. To a vial filled with a 1.0 mL solution (pH ) 4.8) containing 5 mg of TPPTS, 6.5 mg of tricine, 40 mg of mannitol, 38.5 mg of disodium succinate hexahydrate, and 12.7 mg of succinic acid were added 0.2 mL of HYNIC-tetramer solution (100 µg/ mL in water) and 0.3 mL of Na[99mTcO4] solution (370-1850 MBq/mL). The vial was heated at 100 °C for 20-25 min in a lead-shielded water bath. After heating, the vial was placed back into the lead pig and allowed to stand at room temperature for ∼10 min. A sample of the resulting solution was analyzed by the radio-HPLC. The radiochemical purity (RCP) was >95%. The HPLC retention time was ∼15.5 min (Method 2). For biodistribution study, the radiotracer was first prepared and then purified by HPLC. Volatiles in the mobile phases were completely removed under vacuum. Doses were prepared by dissolving the HPLC-purified radiotracer in saline to a concentration of ∼100 µCi/mL. For the blocking experiment, E[c(RGDfK)]2 was dissolved in the solution containing the radiotracer (∼100 µCi/mL) to give a concentration of 7.5 mg/mL. The resulting solution was filtered with a 0.20 µm Millex-LG filter to remove any particles before being injected into animals. Each tumor-bearing mouse was injected with 0.1 mL of solution. Solution Stability. For the stability in kit matrix, samples of the reaction mixture containing the radiotracer were analyzed by HPLC at 0, 2, 4, and 6 h postlabeling. For the cysteine challenging experiment, the solution containing the radiotracer was mixed with an equal volume of a cysteine solution (1 mg/ mL). Samples were analyzed by radio-HPLC at 0, 2, 4, and 6 h. For the solution stability after HPLC purification, the radiotracer was purified by HPLC (Method 2). Volatiles were removed under vacuum. The residue was dissolved in saline. Samples of the resulting solution were analyzed by HPLC at 0, 2, 4, and 6 h postlabeling. Partition Coefficient. The log P value of [99mTc(HYNICtetramer)(tricine)(TPPTS)] was determined using the following procedure: the radiotracer was purified by HPLC. Volatiles were removed under vacuum. The residue was dissolved in a mixture of equal volume (3 mL:3 mL) n-octanol and 25 mM phosphate buffer (pH ) 7.4). After being stirred vigorously for at least 20 min, the mixture was centrifuged at a speed of 8000 rpm for 5 min. Samples (in triplicates) from both n-octanol and aqueous layers were counted in a gamma counter (Beckman Gama 8000). The partition coefficients were calculated. The log P value was measured three different times and reported as an average of three different measurements plus the standard deviation. Cell Integrin Receptor Binding Assay. The in vitro integrinbinding affinity and specificity of HYNIC-tetramer were assessed via displacement cell-binding assays using 125I-echistatin as the integrin-specific radioligand. Experiments were performed on MDA-MB-435 human breast cancer cell line by slight modification of a method previously described (26). In brief, MDA-MB-435 cells were grown in Gibco’s Dulbecco’s medium supplemented with 10% fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 µg/mL streptomycin (Invitrogen Co, Carlsbad, CA), at 37 °C in humidified atmosphere containing 5% CO2. Filter multiscreen DV plates were seeded with 105 cells in binding buffer and incubated with 125I-echistatin in the presence of increasing concentrations of different RGD peptide

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Figure 2. In vitro inhibition of 125I-echistatin binding to Rvβ3 integrin on MDA-MB-435 human breast cancer cells by E[c(RGDfK)]2 (b), E{E[c(RGDfK)]2}2 (O), HYNIC-E[c(RGDfK)]2 (4), and HYNIC-E{E[c(RGDfK)]2}2 (×).

analogues. After the unlabeled 125I-echistatin was removed, hydrophilic PVDF filters were collected and the radioactivity was determined using a gamma counter (Packard, Meriden, CT). The IC50 values were calculated by fitting the data by nonlinear regression using GraphPad Prism (GraphPad Software, Inc., San Diego, CA). Experiments were carried out twice with triplicate samples. The IC50 values are reported as an average of these samples plus the standard deviation. Breast Cancer Xenograft Model. Biodistribution, imaging, and metabolism studies were performed using athymic nude mice bearing MDA-MB-435 human breast cancer xenografts in compliance with NIH animal experiment guidelines (Principles of Laboratory Animal Care, NIH Publication No. 86-23, revised 1985). The animal protocols were approved by the Purdue University Animal Care and Use Committee (PACUC). Female athymic nude (nu/nu) mice were purchased from Harlan (Indianapolis, IN) at 4-5 weeks of age. The mice were implanted with 5 × 106 cells of MDA-MB-435 estrogen receptor-negative human breast cancer cells into the mammary fat pad. When tumors reached 0.4-0.6 cm in mean diameter, the tumor-bearing mice were used to biodistribution and imaging studies. Biodistribution Protocol. Sixteen tumor-bearing mice (2025 g) were anesthetized by intraperitoneal (i.p.) injection of ketamine (40-100 mg/kg) and xylazine (2-5 mg/kg). Once the animal was in surgical plane of anesthesia, as noted by lack of response to pain, the radiotracer (∼1 µCi) dissolved in saline was administered via tail vein. Four tumor-bearing mice were sacrificed by exsanguinations and opening of the thoracic cavity at 5, 30, 60, and 120 min postinjection (p.i.). Blood was withdrawn from the heart through a syringe. Organs of interest (such as tumor, brain, eyes, heart, intestine, kidneys, liver, lungs, muscle, and spleen) were excised, washed with saline, dried with tissue, weighed, and counted on a γ-counter (Beckman RD8000). The organ uptake was calculated as a percentage of the injected dose per gram of wet tissue (%ID/g). For the blocking experiment, four tumor-bearing nude mice (20-25 g) were used, and each animal was administered 0.1 mL saline solution containing ∼1 µCi of the test agent along with ∼750 µg (∼ 30 mg/kg) of E[c(RGDfK)]2. At 1 h p.i., all four animals were sacrificed for organ biodistribution using the same procedure above. Metabolism. Each tumor-bearing mouse was administered with the radiotracer (∼100 µCi/mouse). The urine samples were collected at 2 h p.i. by manual void and were mixed with equal volume of acetonitrile. The mixture was centrifuged at 8000 rpm. The supernatant was collected and filtered through a 0.20 µm Millex-LG syringe filter unit. The filtrate was analyzed by

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Figure 3. Top: Biodistribution data for [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] at 5, 30, 60, and 120 min p.i. in nude mice bearing the MDAMB-435 human breast cancer xenografts; Bottom: Comparison of the organ uptake (% ID/g) for [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] at 60 min p.i. in the absence/presence of excess E[c(RGDfK)]2. Each data point represents an average of biodistribution data in four animals.

radio-HPLC (Method 2). The feces samples were collected at 2 h p.i. and suspended in a mixture of 50% acetonitrile aqueous solution, and the resulting mixture was vortexed for 5-10 min. After centrifugation at 8000 rpm for 5 min, the supernatant was collected and passed through a 0.20 µm Millex-LG syringe driven filter unit. The filtrate was analyzed by radio-HPLC. Two tumor-bearing mice were used for the metabolism study. SPECT Imaging. Imaging studies were performed by Dr. Wang’s group using athymic nude mice bearing MDA-MB435 breast cancer xenografts in accordance with guidelines of Peking University Health Science Center Animal Care and Use Committee. Animals were anesthetized with i.p. injection of sodium pentobarbital at a dose of 45.0 mg/kg. Each animal was administered ∼400 µCi of the radiotracer in 0.2 mL of saline. Animals were placed prone on a dual head γ-camera (Siemens, E. CAM) equipped with a parallel-hole, low-energy, and highresolution collimator. Anterior static images were acquired at 1, 2, and 4 h p.i. and were stored digitally in a 128 × 128 matrix. The acquisition count limits were set at 1000 K. After completion of imaging, animals were sacrificed by cervical dislocation.

Statistical Analysis. The biodistribution data and T/B ratios were reported as an average plus the standard variation based on the results from four animals for each time point. Comparison between two different radiotracers was also made using the oneway ANOVA test. The level of significance was set at P < 0.05.

RESULTS Synthesis of HYNIC-Tetramer. The HYNIC-tetramer conjugate was prepared by coupling E{E[c(RGDfK)]2}2 with HYNIC-NHS under slightly basic condition (pH ) 8.5-9.0) in a mixture of water and DMF (50:50 ) v:v). It was purified by HPLC (Method 1). Its identity has been confirmed by ESIMS data (m/z ) 3050.93 for [M + H]+ and 1526.89 for [M + 2H]2+). Integrin Rvβ3 Binding. The integrin Rvβ3-positive MDAMB-435 breast cancer cells were used for the in vitro binding affinity studies instead of the immobilized integrin Rvβ3. We compared the integrin Rvβ3 binding affinity of HYNIC-tetramer, HYNIC-dimer, E[c(RGDfK)]2, and E{E[c(RGDfK)]2}2 by

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Figure 4. Direct comparison of organ uptake (% ID/g) between [99mTc(HYNIC-dimer)(tricine)(TPPTS)]) and [99mTc(HYNIC-tetramer)(tricine)(TPPTS)]) in athymic nude mice bearing MDA-MB-435 human breast cancer xenografts.

competitive displacement of 125I-echistatin. Both cyclic RGDfK peptides and their HYNIC conjugates inhibited the binding of 125I-echistatin to MDA-MB-435 breast cancer cells (Figure 2). The IC50 values for E{E[c(RGDfK)]2}2 and E[c(RGDfK)]2 were 51 ( 11 nM and 78 ( 27 nM, respectively. IC50 values for HYNIC-tetramer and HYNIC-dimer were 55 ( 11 nM and 52 ( 9 nM under the same condition. E{E[c(RGDfK)]2}2 has a slightly higher integrin Rvβ3 binding affinity than E[c(RGDfK)]2. There was no significant difference in the integrin Rvβ3 binding affinity between their HYNIC conjugates within the experimental error. Attachment of HYNIC did not alter the binding affinity of the tetramer E{E[c(RGDfK)]2}2. Radiochemistry. [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] was prepared using a non-stannous formulation since TPPTS is a strong reducing agent for Na[99mTcO4] (33). The RCP was >95% with less than 0.5% of [99mTc]colloid. [99mTc(HYNICtetramer)(tricine)(TPPTS)] was analyzed using a reversed phase radio-HPLC method (Method 2). The log P value for [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] was -3.29 ( 0.22 in a mixture of n-octanol and 25 mM phosphate buffer, pH ) 7.4. This value is lower than that (-2.66 ( 0.25) of its dimeric analogue [99mTc(HYNIC-dimer)(tricine)(TPPTS)], suggesting that increasing the peptide multiplicity decreases lipophilicity of the 99mTc-labeled cyclic RGDfK peptides. The solution stability of [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] was monitored by radio-HPLC (Method 2). It was found that it remained stable in the kit matrix and in presence of cysteine challenge (1 mg/mL) for >12 h. It was also stable for >6 h after purification.

Biodistribution. [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] was purified by HPLC to remove the “unlabeled” HYNICtetramer. Biodistribution was performed using the athymic nude mice bearing MDA-MB-435 human breast cancer xenografts. Figure 3 showed its biodistribution data in the selected organs with/without the co-injection of excess E[c(RGDfK)]2. The organ uptake was expressed as %ID/g. Each data point represents an average in four animals. Figures 4 and 5 illustrate the comparison of organ uptake and T/B ratios between [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] and [99mTc(HYNICdimer)(tricine)(TPPTS)] in several selected organs, such as tumor, blood, liver, lungs, and kidneys. Biodistribution data and T/B ratios of [99mTc(HYNIC-dimer)(tricine)(TPPTS)] were obtained from our previous study (24). In general, [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] displayed a rapid clearance predominantly via renal route. The tumor uptake of [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] (5.60 ( 0.87 %ID/g and 7.30 ( 1.32 %ID/g at 5 and 120 min p.i., respectively) was significantly higher than that of [99mTc(HYNIC-dimer)(tricine)(TPPTS)] (3.50 ( 0.18 %ID/g and 3.82 ( 0.32 %ID/g at 5 and 120 min p.i., respectively) over the 2 h study period. The kidney uptake of [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] (33.05 ( 5.75 %ID/g and 25.93 ( 2.52 %ID/g at 5 and 120 min p.i., respectively) was much higher than that of [99mTc(HYNIC-dimer)(tricine)(TPPTS)] (24.15 ( 1.23 %ID/g and 14.33 ( 2.39 %ID/g at 5 and 120 min p.i., respectively) during the same study period. The blood radioactivity level for [99mTc(HYNIC-tetramer)(tricine)(TPPTS)]

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Figure 5. Direct comparison of T/B ratios between [99mTc(HYNICdimer)(tricine)(TPPTS)]) and [99mTc(HYNIC-tetramer)(tricine)(TPPTS)]) in athymic nude mice bearing MDA-MB-435 human breast cancer xenografts.

(4.61 ( 0.82 %ID/g) was significantly (p < 0.01) higher than that of [99mTc(HYNIC-dimer)(tricine)(TPPTS)] (2.70 ( 0.12 %ID/g) at 5 min p.i.; but this difference disappeared at 30-120 min p.i. (Figure 4). A similar trend was observed for their liver uptake. As a result, [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] has the tumor/blood, tumor/liver and tumor/ lung ratios that are significantly better (p < 0.05) than those of [99mTc(HYNIC-dimer)(tricine)(TPPTS)] at 30-120 min p.i. (Figure 5). Blocking Experiment. In this experiment, E[c(RGDfK)]2 (IC50 ) 78.27 ( 27.59 nM against 125I-echistatin binding to the integrin Rvβ3-positive MDA-MB-435 breast cancer cells) was used as the blocking agent at a dose of ∼30 mg/kg (∼750 µg per mouse). Such a high dose was used to make sure that the integrin Rvβ3 is almost completely blocked. It was found that co-injection of a large excess of E[c(RGDfK)]2 resulted in a significant reduction of the uptake of [99mTc(HYNIC-tetramer))(tricine)(TPPTS)] in the tumor. In addition, there was also substantial decrease of the uptake for [99mTc(HYNICtetramer))(tricine)(TPPTS)] in lungs, liver, spleen, and intestine (Figure 3). The muscle and kidney uptake was also inhibited (to a lesser degree). The blood activity in the presence of E[c(RGDfK)]2 was higher than that without E[c(RGDfK)]2.

Figure 6. Radio-HPLC chromatograms of [99mTc(HYNIC-dimer)(tricine)(TPPTS)] in the kit matrix before injection, in urine and feces at 120 min p.i. Each tumor-bearing mouse was administered with ∼3.7 MBq of radiotracer.

Metabolism. The metabolism study on [99mTc(HYNICtetramer)(tricine)(TPPTS)] was performed using the tumorbearing mice. Since it is excreted via both renal and hepatobiliary routes, we collected samples from urine and feces to determine if they are able to retain their chemical integrity at 2 h postinjection. Figure 6 sowed typical radio-HPLC chromatograms of [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] in the kit matrix, urine, and feces at 120 min postinjection. About 80% metabolite of [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] was found in the urine while only ∼15% metabolism was detected in the feces sample at 2 h p.i. No efforts have been made to identify the metabolite(s). SPECT Imaging. Figure 7 illustrated the selected SPECT images of tumor-bearing mice administered with ∼15 MBq of [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] at 1, 2, and 4 h postinjection. Tumors were clearly visualized in all three SPECT images. The activity level in the abdominal region (particularly liver and lungs) was very low, which agreed well with the ex vivo biodistribution data at the same time point (Figure 4).

DISCUSSION The in vivo distribution pattern of a 99mTc-based radiotracer is determined by several factors, including binding affinity of the targeting biomolecule, internalization of the radiotracer, Tc chelate, molecular charge, lipophilicity, and metabolic stability.

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Figure 7. Representative static SPECT images of the tumor-bearing mice administered with ∼15 MBq of [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] and at 1, 2, and 4 h p.i. Arrows indicate the presence of tumors.

Since the natural mode of interactions between integrin Rvβ3 and RGD-containing proteins may involve multivalent binding sites, the idea to improve integrin Rvβ3 binding affinity with multivalent RGD peptides could provide more effective antagonists with better targeting capability and higher cellular uptake through the integrin-dependent endocytosis pathway (35). Several groups applied the multivalent concept to develop integrin Rvβ3-targeted radiotracers. Rajopadhye et al. were the first to use cyclic RGD peptide dimers, such as E[c(RGDfK)]2, as targeting biomolecules for development of diagnostic (99mTc and 111In) and therapeutic (90Y) radiotracers (37, 38). We also used E[c(RGDyK)]2 to prepare 64Cu-based PET radiotracers (26). Kessler et al. reported the hexaethylene glycolic acid (HEG) and poly(lysine)-tethered cyclic RGDfE dimers and tetramers with better integrin Rvβ3 binding affinity than its monomeric analogues (30, 31). The HEG and poly(lysine) linkers were used to increase the distance between RGD motifs. It was found that the minimum distance is about 3.5 nm (∼25 bond distances) for simultaneous binding of two RGD motifs in the immobilized integrin Rvβ3 assay (30, 31). The success of E[c(RGDfK)]2 and E[c(RGDyK)]2 as targeting biomolecules for the integrin Rvβ3 radiotracers is very intriguing. Given the short distance between two cyclic RGD motifs, it is unlikely that they would bind to the adjacent integrin Rvβ3 receptors simultaneously. However, the binding of one RGD motif to integrin Rvβ3 will significantly increase “local concentration” of the second RGD motif in the vicinity of receptor site. This might lead to the enhanced integrin Rvβ3 binding rate or reduced dissociation rate of RGD peptide from integrin Rvβ3. The high “local RGD peptide concentration” may explain the higher tumor uptake and longer tumor retention times of the radiolabeled (99mTc, 111In, and 64Cu) cyclic RGD dimers as compared to their monomeric analogues (26, 37, 38). The longest distance between RGD motifs of E{E[c(RGDfK)]2}2 is ∼30 bond lengths. Its integrin Rvβ3 binding affinity (IC50 ) 15 ( 1 nM) is higher than that of E[c(RGDfK)]2 (IC50 ) 32 ( 2 nM) against 125I-echistatin in binding to the integrin Rvβ3-positive U87MG glioma cancer cells (27). In this study, the integrin Rvβ3 binding affinity of E{E[c(RGDfK)]2}2 (IC50 ) 51 ( 11 nM) is only slightly higher than that of E[c(RGDfK)]2 (IC50 ) 78 ( 27 nM) against 125I-echistatin binding to the integrin Rvβ3-positive MDA-MB-435 breast

cancer cells. There is no significant difference in IC50 values between HYNIC-tetramer (55 ( 11 nM) and HYNIC-dimer (52 ( 9 nM). We believe that the different results obtained from two in vitro assays might be related to the integrin Rvβ3 expression level on tumor cells (glioma versus breast cancer). Recently, we have demonstrated that the integrin Rvβ3 density on MDA-MB-435 breast cancer cells was not as high as that on U87MG glioma cancer cells (39). Therefore, the distance between the two neighboring integrin Rvβ3 receptors would be longer, which makes it more difficult for two RGD motifs in E{E[c(RGDfK)]2}2 and E[c(RGDfK)]2 to bind to the adjacent integrin Rvβ3 simultaneously. This might explain the fact that the IC50 of HYNIC-tetramer (55 ( 11 nM) using MDAMB-435 breast cancer cells is more than 3-fold higher than that (15 ( 1 nM) using U87MG glioma cancer cells. This may also explain why the IC50 values for RGD peptides (monomers, dimers, and tetramers) obtained with the immobilized integrin Rvβ3 assay (26-29) are always much lower that those obtained using the whole-cell integrin Rvβ3-binding assay (30, 31). The in vivo tumor targeting capability of [99mTc(HYNICtetramer)(tricine)(TPPTS)] was evaluated in athymic nude mice bearing MDA-MB-435 human breast cancer xenografts. It was found to have a much higher tumor uptake than its dimeric analogue over the 2 h study period, even though HYNICtetramer has the IC50 (55 ( 11 nM) similar to that of HYNICdimer (52 ( 9 nM). Among the radiotracers evaluated in this animal model (24, 25), [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] has the highest tumor uptake (7.30 ( 1.32 %ID/g) at 2 h p.i. One might argue that the enhanced tumor uptake is related to higher molecular weight of the cyclic RGDfK tetramer, resulting in longer blood circulation time. However, there is no significant difference in the blood activity levels and the liver uptake between [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] and [99mTc(HYNIC-dimer)(tricine)(TPPTS)] at 30-120 min p.i. The tumor/blood ratio of [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] (13.15 ( 1.45) is almost twice of that of [99mTc(HYNIC-dimer)(tricine)(TPPTS)] (7.32 ( 0.44) at 120 min p.i. (Figure 5). The tumor/liver and tumor/lung ratios of [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] are also significantly better (p < 0.05) than those of [99mTc(HYNIC-dimer)(tricine)(TPPTS)] at 30-120 min

99mTc-Labeled

Cyclic RGD Tetramer

p.i. Clearly, the tetramer E{E[c(RGDfK)]2}2 is a better integrin Rvβ3-targeting biomolecule than its monomeric and dimeric analogues. The kidney uptake of [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] is significantly higher than that of its dimeric analogue at all four time points (Figure 4). Small peptides in blood plasma are filtered through glomerular capillaries in the kidneys and subsequently reabsorbed by proximal tubular cells by carriermediated endocytosis. Membranes of renal tabular cells contain negatively charged sites to which the positively charged groups, such as guanidine moieties in cyclic RGD peptides, are expected to bind. In this respect, one might suggest that different charges of E{E[c(RGDfK)]2}2 and E[c(RGDfK)]2 could cause the difference in kidney uptake of [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] and [99mTc(HYNIC-dimer)(tricine)(TPPTS)] because E{E[c(RGDfK)]2}2 has more positively charged R-residues under physiological conditions. It has been shown that positively charged peptides are more efficiently reabsorbed by the proximal renal tubular cell than neutral peptides (40). The high kidney uptake and prolonged kidney retention has also been attributed to positively charged K residues of 99mTc-DKCK-c(RGDfK), in which one K residue is part of the Tc chelate and one is used as a linker between c(RGDfK) and the Tc chelate in addition to the K residue in c(RGDfK) (40). However, this explanation cannot account for the fact that the kidney uptake of [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] can be partially blocked by excess E[c(RGDfK)]2 (Figure 3). Therefore, the high kidney uptake and prolonged kidney retention of [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] is probably caused by several factors, including the 99mTc chelate (99mTc-HYNICtricine-TPPTS), presence of more R-residues, integrin Rvβ3binding, high molecular weight of E{E[c(RGDfK)]2}2. The tumor-specificity of [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] was demonstrated by co-injecting excess E[c(RGDfK)]2, which resulted in significant reduction in the tumor uptake of [99mTc(HYNIC-tetramer))(tricine)(TPPTS)]. Since the integrin Rvβ3 expression in lungs and liver of rats has been documented (41), it is not surprising to see a high uptake in liver and lungs of tumor-bearing mice. The blood activity of [99mTc(HYNICtetramer))(tricine)(TPPTS)] with excess E[c(RGDfK)]2 was higher than that without E[c(RGDfK)]2, is probably caused by its saturation effect. Similar results were obtained for [99mTc(HYNIC-dimer)(tricine)(TPPTS)] and 111In-DOTA-E[c(RGDfK)]2 in the BALB/c mice bearing ovarian carcinoma (28, 29), 64Cu-DOTA-E[c(RGDfK)] in nude mice bearing the MDA2 MB-435 breast cancer xenografts (26), and 64Cu-DOTA-E{E[c(RGDfK)]2}2 in nude mice bearing UG87MG glioma xenografts (27). Radiolabeled cyclic RGD peptides (monomers, dimers, and tetramers) often accumulate in kidneys. Extensive metabolism has been detected for the 99mTc-labeled RGDfK monomer (39) and 64Cu-labeled cyclic RGDfK tetramer in kidneys and urine samples (27). In this study, only 20% of [99mTc(HYNICtetramer)(tricine)(TPPTS)] remained intact in urine at 120 min p.i. Similar results were observed for [99mTc(HYNIC-dimer)(tricine)(TPPTS)] (24). Surprisingly, >85% of [99mTc(HYNICtetramer)(tricine)(TPPTS)] remains intact in the feces sample at 2 h p.i. It is possible that the metabolized species can be reabsorbed and then excreted via renal system.

CONCLUSION In summary, we studied biodistribution properties of the 99mTc-labeled RGD tetramer, [99mTc(HYNIC-tetramer)(tricine)(TPPTS)], in athymic nude mice bearing MDA-MB-435 human breast cancer xenografts. The key finding of this study is that the tetramer is a much better integrin Rvβ3-targeting biomolecule than its monomeric and dimeric analogues. The peptide

Bioconjugate Chem., Vol. 18, No. 2, 2007 445

multiplicity has a significant impact on biodistribution characteristics and metabolic patterns of 99mTc-labeled cyclic RGD peptides. On the basis of tumor uptake and T/B (particularly tumor/liver and tumor/lung) ratios, we believe that [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] is a promising new radiotracer for noninvasive imaging of the integrin Rvβ3-positive tumors by SPECT.

ACKNOWLEDGMENT The authors would like to thank Lee Ann Grote, Carol Dowell, and Cheryl Anderson from the Department of Veterinary Clinical Sciences, Purdue University, for their assistance with biodistribution studies. This work is supported in part by research grants: 1R01 CA115883-01A2 (S.L. and C.X.) from the National Cancer Institute (NCI) and BCTR0503947 (S.L.) from the Susan G. Komen Breast Cancer Foundation. Supporting Information Available: Additional experimental data. This material is available free of charge via the Internet at http://pubs.acs.org.

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