99mTc-Labeled Cyclic RGDfK Dimer: Initial Evaluation for SPECT

Bioconjugate Chem. 2006, 17, 1069−1076

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Cyclic RGDfK Dimer: Initial Evaluation for SPECT Imaging of Glioma Integrin rvβ3 Expression Bing Jia,†,| Jiyun Shi,†,| Zhi Yang,§ Bing Xu,§ Zhaofei Liu,† Huiyun Zhao,† Shuang Liu,‡,* and Fan Wang†,* Medical Isotopes Research Center, Peking University, Beijing 100083, China, Department of Isotopes, China Institute of Atomic Energy, Beijing 102413, China, School of Health Sciences, Purdue University, Indiana 47907, and Department of Nuclear Medicine, Peking University Oncology School, Beijing 100036, China. Received March 5, 2006; Revised Manuscript Received April 24, 2006

This report describes the evaluation of biodistribution properties of three radiotracers, [99mTc(SQ168)(EDDA)], [99mTc(SQ168)(tricine)(PDA)], and [99mTc(SQ168)(tricine)(TPPTS)] (SQ168 ) [2-[[[5-[carboonyl]-2-pyridinyl]hydrazono]methyl]benzenesulfonic acid]-Glu(cyclo{Lys-Arg-Gly-Asp-D-Phe})-cyclo{Lys-Arg-Gly-Asp-D-Phe}; EDDA ) ethylenediamine-N,N′-diacetic acid; PDA ) 2,5-pyridinedicarboxylic acid; TPPTS ) trisodium triphenylphosphine-3,3′,3′′-trisulfonate), and their potential to image the glioma integrin Rvβ3 expression in BALB/c nude mice bearing the U87MG human glioma xenografts. It was found that all three radiotracers were able to localize in glioma tumors with a relatively high tumor uptake and long tumor retention time by binding to the integrin Rvβ3 expressed on both tumor cells and endothelial cells of tumor neovasculature. It seems that the coligand has minimal effect on integrin Rvβ3 targeting capability of the 99mTc-labeled RGDfK dimer, but it has a significant impact on their biodistribution properties. For example, the complex [99mTc(SQ168)(tricine)(TPPTS)] has the lowest liver uptake and the highest metabolic stability in normal BALB/c nude mice. Results from SPECT imaging studies show that the glioma tumors can be clearly visualized with all three radiotracers at 4 h postinjection. Among the three radiotracers evaluated in this study, [99mTc(SQ168)(tricine)(TPPTS)] has the best imaging quality and is a promising candidate for more preclinical evaluations in the future.

INTRODUCTION Gliomas or tumors derived from glial cells of the central nerve system are the most frequent primary intracranial neoplasma in adults. Malignant gliomas are the second leading cause of cancer death in children under age 15 and also in young adults up to age 34 (1, 2). With a high proliferation rate, marked neovascularization, and extensive local invasion of tumor cells into the normal brain parenchyma, gliomas are resistant to conventional treatment, such as surgery, chemotherapy, and radiation therapy. The median survival of aggressively treated patients with glioblastoma is about 12 months (3). Therefore, early detection of brain tumors such as glioma is highly desirable so that various therapeutic regimens can be given before the primary tumors become widely spread. Since gliomas are extremely invasive with a high level of integrin Rvβ3 overexpression (4-6), which also correlates with metastatic disease human neuroblastomas (7), it would be of great clinical benefit to develop a radiotracer that can be used not only to noninvasively visualize and quantify integrin Rvβ3 expression but also to select patients under consideration for anti-integrin treatment and monitor treatment efficacy in integrin Rvβ3-positive patients. For the last several years, many high affinity integrin Rvβ3 antagonists (RGD-containing cyclic peptides and nonpeptide RGD mimetics) have been proposed as targeting biomolecules * To whom correspondence should be addressed. Dr. Shuang Liu, School of Health Sciences, Purdue University, 550 Stadium Mall Drive, West Lafayette, IN 47907. Phone: 765-494-0236, Fax: 765-496-3367, E-mail: [email protected]; or Dr. Fan Wang, Medical Isotopes Research Center, Peking University, 38# Xueyuan Road, Beijing 100083, China. Phone and Fax: 86-10-82801145, E-mail: [email protected]. † Peking University. ‡ Purdue University. § Peking University Oncology School. | China Institute of Atomic Energy.

to carry the diagnostic “probes” into the integrin Rvβ3-positive tumors (8-13). Several molecular probes have been developed for magnetic resonance imaging (14), ultrasound (15), optical imaging (16), positron emission tomography (PET) (17-23), and single-photon emission computed tomography (SPECT) imaging (24-28). Although most of “molecular” probes containing a monomeric RGD-tripeptide sequence are able to target tumors by binding to the integrin Rvβ3 overexpressed on tumor cells and endothelial cells of the tumor neovasculature, they suffer from several shortcomings, such as low tumor uptake, which limit their application as tumor imaging agents in clinics. We and others have been using cyclic RGD peptide dimers, such as E[c(RGDfK)]2, for development of the integrin Rvβ3targeted diagnostic (64Cu, 99mTc, and 111In) radiotracers (2935). Recently, we reported evaluation of complexes [99mTc(SQ168)(tricine)(L)] (Figure 1: SQ168 ) [2-[[[5-[carboonyl]-2-pyridinyl]hydrazono]methyl]benzenesulfonic acid]-Glu(cyclo{LysArg-Gly-Asp-D-Phe})-cyclo{Lys-Arg-Gly-Asp-D-Phe}; L ) ISONIC (isonicotinic acid), TPPTS (trisodium triphenylphosphine-3,3′,3′′-trisulfonate), and PDA (2,5-pyridinedicarboxylic acid)) as radiotracers for imaging the integrin Rvβ3 expression in nude mice bearing the MDA-MB-435 human breast cancer xenografts (29, 31). It was found that coligands have minimal effect on tumor targeting capability, but it has a significant impact on excretion kinetics of the 99mTc-labeled cyclic RGDfK dimer. Results from the blocking experiment suggest that the tumor localization of the 99mTc-labeled cyclic RGDfK dimer is indeed integrin Rvβ3-mediated (31). As continuation of our interest in the integrin Rvβ3-targeted radiotracers for imaging solid tumors of different origin, we have evaluated the biodistribution characteristics of ternary ligand complexes [99mTc(SQ168)(tricine)(PDA)] and [99mTc(SQ168)(tricine)(TPPTS)] in the female BALB/c nude mice bearing the U87MG human glioma xenografts. The main objective is to assess their potential as SPECT radiotracers for imaging the integrin Rvβ3-expression in gliomas. For comparison

10.1021/bc060055b CCC: $33.50 © 2006 American Chemical Society Published on Web 06/29/2006

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Figure 1. Structures of the HYNIC-conjugated cyclic RGDfK dimer (SQ168), coligands, and their ternary ligand complexes [99mTc(SQ168)(tricine)(L)] (SQ168 ) HYNIC-E[c(RGDfK)]2; L ) TPPTS, ISONIC, NIC, and PDA).

purposes, we also evaluated the complex [99mTc(SQ168)(EDDA)] (EDDA ) ethylenediamine-N,N′-diacetic acid) in the same tumor animal model. EDDA has been used as a coligand for preparation of 99mTc-labeled somatostatin analogues, which are under investigation as new radiotracers for imaging somatostatin receptor-positive tumors in humans (36-38).

EXPERIMENTAL SECTION Materials and Methods. All chemicals were purchased from Aldrich Chemical Co., unless specified. Synthesis of SQ168 (HYNIC-E[c(RGDfK)]2) was described in our previous communication (31). Na99mTcO4 was obtained from a commercial 99Mo/99mTc generator (Beijing Atom High Tech Co., Ltd.). The radio-HPLC method used a HP Hewlett Packard Series 1100 HPLC system equipped with Radioflow Detector LB509 and Agilent Zorbax SB-C18 column (4.6 mm × 250 mm, 5 µm). The flow rate was 1.0 mL/min. The mobile phase was isocratic with 90% solvent A (25 mM NaOAc 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, then to 10% solvent B at 25 min. Synthesis of [99mTc(SQ168)(EDDA)]. To a clean vial were added 100 µL of SQ168 solution (100 µg/mL in H2O), 100 µL of tricine solution (100 mg/mL in 25 mM succinate buffer, pH 5.0), 20 µL of SnCl2 solution (3.0 mg/mL in 0.1 N HCl), and 100 µL of Na99mTcO4 solution (∼10 mCi). The reaction mixture was kept at room temperature for 10 min. To the solution was added 200 µL of EDDA (30 mg/mL in 1 M NaOH, pH 7.0). The mixture was heated at 100 °C for 30 min. After radiolabeling, a sample of the resulting solution was analyzed by radioHPLC. Synthesis of [99mTc(SQ168)(tricine)(L)] (L ) PDA and TPPTS). To a clean vial were added 100 µL of SQ168 solution (100 µg/mL in H2O), 100 µL of tricine solution (100 mg/mL in 25 mM succinate buffer, pH 5.0), 20 µL of SnCl2 solution (3.0 mg/mL in 0.1 N HCl), and 100 µL of Na99mTcO4 (∼10

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mCi). The reaction mixture was kept at room temperature for 10 min. To the solution above was added 120 µL of TPPTS or PDA solution (50 mg/mL in 25 mM succinate buffer, pH 5.0), and the mixture was heated at 100 °C for 30 min. After radiolabeling, a sample of the resulting solution was analyzed by radio-HPLC. Doses Preparation for Animal Studies. All three 99mTc radiotracers were purified using Sep-Pak C-18 cartridge (Waters, Milford MA) before animal studies. The Sep-Pak C-18 cartridge was activated with 10 mL of ethanol followed by washing with 10 mL of water. The radiotracer was passed through a Sep-Pak C-18 cartridge to filter out the [99mTc]-colloid. The Sep-Pak C-18 cartridge was then washed with 10 mL of saline to remove unlabeled SQ168 and excess coligand. The 99mTc radiotracer was eluted with 0.4 mL of 80% ethanol. Doses for animal studies were prepared by dissolving the purified radiotracer in saline to give a concentration of 150 µCi/mL for biodistribution studies, 4.0 mCi/mL for metabolism, and 2.0 mCi/mL for imaging studies. For the blocking experiment, E[c(RGDfK)]2 was used as the blocking agent. Doses were prepared by dissolving E[c(RGDfK)]2 in saline to give a final concentration of 3.0 mg/mL. Each tumor-bearing mouse was injected with 0.1 mL of E[c(RGDfK)]2 solution, which corresponds to a dose of 15 mg/kg. Animal Studies. Both biodistribution and imaging studies were performed by Dr. Wang’s group at the Peking University Medical Isotopes Research Center, according to the literature (21, 23, 39). All animal experiments were performed in accordance with guidelines of Peking University Health Science Center Animal Care and Use Committee. Tumor-Bearing Mice. The human U87MG glioma tumor cells from Professor Jingde Zhu (Shanghai Cancer Research Institute) were maintained at 37 °C and 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS). Female BALB/c nude mice (4-5 weeks of age) were purchased from Department of Animal Experiment, Peking University Health Science Center. The U87MG cells (5 × 106) were implanted subcutaneously into the right upper flanks of mice. When tumors reached ∼0.8 cm in mean diameter, the tumor-bearing mice were used to biodistribution and imaging studies. Biodistribution. Sixteen tumor-bearing mice were randomly divided into four groups, each of which had four animals. Animals were anesthetized with an intraperitoneal injection of sodium pentobarbital at a dose of 45.0 mg/kg. The 99mTc radiotracer (∼15 µCi in 0.1 mL saline) was administered intravenously to each tumor-bearing mouse. Animals were sacrificed by cervical dislocation at 0.5, 1, 2, and 4 h postinjection. Blood, heart, liver, spleen, kidney, stomach, intestine, muscle, and tumor were harvested, weighed, and measured for radioactivity in a γ-counter (Wallac 1470-002, Perkin-Elmer, Finland). The organ uptake was calculated as a percentage of the injected dose per gram of wet tissue mass (%ID/g). For the blocking experiment, each animal was administered with 300 µg of E[c(RGDfK)]2 (0.1 mL in saline) 30 min prior to the injection of [99mTc(SQ168)(tricine)(PDA)]. At 1 h postinjection, all four animals were sacrificed for biodistribution using the same procedure above. The results were compared to those obtained in animals without the injection of E[c(RGDfK)]2. Metabolism. The metabolic stability of 99mTc radiotracers was evaluated in normal BALB/c nude mice. Animals were anesthetized with intraperitoneal injection of sodium pentobarbital (45.0 mg/kg). Each mouse was administered with the radiotracer at a dose of 800 µCi in 0.2 mL saline via intravenous injection. The mice were sacrificed by cervical dislocation at 2 h postinjection, and livers were removed and homogenized. The homogenate was extracted with 500 µL of 50% acetonitrile aqueous solution. The extract was centrifuged at 1500 rpm for

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Statistical Analysis. The biodistribution data and T/B ratios are reported as an average plus the standard variation. Comparison between two different radiotracers was also made using the one-way ANOVA test. The level of significance was set at p ) 0.05.

RESULTS

Figure 2. Solution stability data for [99mTc(SQ168)(EDDA)], [99mTc(SQ168)(tricine)(PDA)], and [99mTc(SQ168)(tricine)(TPPTS)] in the presence of cysteine (1.0 mg/mL).

15 min, and the supernatant was filtered through a 0.22 µ MillexLG syringe driven filter unit. The filtrate was analyzed by radioHPLC. Urine samples were collected at 2 h postinjection and were mixed with equal volume of acetonitrile. The mixtures were centrifuged at 1500 rpm for 15 min. The supernatants were collected and filtered through a 0.22 µ Millex-LG syringe driven filter unit. The filtrates were analyzed by radio-HPLC. SPECT Imaging. SPECT imaging was performed on nine tumor-bearing mice (three groups). Animals were anesthetized with an intraperitoneal injection of sodium pentobarbital at a dose of 45.0 mg/kg. Each animal was administered with 400 µCi of the 99mTc radiotracer in 0.2 mL of saline. Animals were placed supine on a two-head γ-camera (SIEMENS, E. CAM) equipped with a parallel-hole, low-energy, and high-resolution collimator. Anterior images were acquired 4 h postinjection and 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.

Radiochemistry. Complexes [99mTc(SQ168)(EDDA)], [99mTc(SQ168)(tricine)(PDA)], and [99mTc(SQ168)(tricine)(TPPTS)] were prepared using a two-step radiolabeling procedure. First, HYNIC-E[c(RGDfK)]2 (SQ168) was allowed to react with Na99mTcO4 in the presence of excess tricine and stannous chloride to form the [99mTc(SQ168)(tricine)n] intermediate, which underwent ligand exchange with EDDA to give the complex [99mTc(SQ168)(EDDA)], or reacted with PDA or TPPTS to form ternary ligand complexes [99mTc(SQ168)(tricine)(L)] (L ) PDA and TPPTS). The radiochemistry purity (RCP) for all three 99mTc radiotracers was >95% after purification with the Sep-Pak C-18 cartridge before animal studies. Solution Stability. The solution stability of complexes [99mTc(SQ168)(EDDA)], [99mTc(SQ168)(tricine)(PDA)], and [99mTc(SQ168)(tricine)(TPPTS)] was monitored by radio-HPLC after addition of cysteine. Figure 2 shows their solution stability data. It is quite clear that all three radiotracers remain stable over 6 h in the presence of cysteine (1.0 mg/mL). Biodistribution. Biodistribution studies were performed using female BALB/c nude mice bearing the U87MG human glioma xenografts. This animal model has been used to evaluate radiolabeled RGD peptides as potential radiotracers for imaging gliomas (21, 23, 39). The selected biodistribution data for [99mTc(SQ168)(EDDA)], [99mTc(SQ168)(tricine)(PDA)], and [99mTc(SQ168)(tricine)(TPPTS)] with/without the presence of excess E[c(RGDfK)]2 are summarized in Figure 3. Figure 4 illustrates their tumor-to-background (T/B) ratios in several selected organs. Figure 5 shows the direct comparison of their organ uptake at 0.5, 1, 2, and 4 h postinjection and excretion kinetics from the blood, liver, and kidneys.

Figure 3. Comparison of the organ uptake (%ID/g) between [99mTc(SQ168)(EDDA)], [99mTc(SQ168)(tricine)(PDA)], and [99mTc(SQ168)(tricine)(TPPTS)] in BALB/c nude mice bearing the U87MG human glioma xenografts. Each data point represents an average of biodistribution data in four animals.

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Figure 4. Tumor/background ratios for [99mTc(SQ168)(EDDA)], [99mTc(SQ168)(tricine)(PDA)], and [99mTc(SQ168)(tricine)(TPPTS)] in BALB/c nude mice bearing the U87MG human glioma xenografts at 4 h postinjection.

In general, all three complexes, [99mTc(SQ168)(EDDA)], [99mTc(SQ168)(tricine)(PDA)], and [99mTc(SQ168)(tricine)(TPPTS)], displayed a rapid clearance from the blood via renal and hepatobiliary routes. Their blood clearance curves were almost identical (Figure 5), suggesting that coligands have little impact on their protein binding. Their tumor uptake remained relatively unchanged over 2 h within the experimental error (Figure 5). However, the tumor uptake of [99mTc(SQ168)(tricine)(PDA)] (3.01 ( 0.47%ID/g) and [99mTc(SQ168)(tricine)(TPPTS)] (3.10(0.35%ID/g) was significantly (p < 0.01) higher than that of [99mTc(SQ168)(EDDA)] (1.99 ( 0.44%ID/g) at 4 h postinjection. The kidney uptake of [99mTc(SQ168)(tricine)(PDA)] and [99mTc(SQ168)(tricine)(TPPTS)] was higher than that of [99mTc(SQ168)(EDDA)]. The liver uptake of [99mTc(SQ168)(tricine)(TPPTS)] was lower than that of [99mTc(SQ168)(EDDA)] and [99mTc(SQ168)(tricine)(PDA)] at >1 h postinjection (Figure 5). As a result, it has the best tumor-toliver ratio (2.39 ( 0.46) at 4 h postinjection (Figure 4). The blocking experiment was performed by preinjecting or coinjecting a known receptor ligand to demonstrate the tumorspecificity of target-specific radiotracers. In this study, we used E[c(RGDfK)]2 (15 mg/kg or 300 µg/mouse), the IC50 of which was 32.1 ( 2.1 nM against 125I-echistatin binding to integrin Rvβ3 on U87MG tumor cells (39), as the blocking agent. Figure 3 shows the comparison of uptake (%ID/g) for [99mTc(SQ168))(tricine)(PDA)] at 1 h postinjection with/without E[c(RGDfK)]2. Obviously, excess E[c(RGDfK)]2 resulted in a significant

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reduction in the uptake of [99mTc(SQ168))(tricine)(PDA)] in the tumor, lungs, liver, spleen, stomach, and guts. The kidney uptake was also inhibited (to a lesser degree) by excess E[c(RGDfK)]2 (Figure 3). Similar results were obtained for [99mTc(SQ168)(tricine)(TPPTS)] and 111In-DOTA-E[c(RGDfK)]2 in the BALB/c mice bearing ovarian carcinoma (33-35) and for [99mTc(SQ168)(tricine)(ISONIC)] and 64Cu-DOTA-E[c(RGDfK)]2 in the nude mice bearing the MDA-MB-435 human breast cancer xenografts (31, 32). However, there is no significant difference in the blood activity with/without excess E[c(RGDfK)]2 (Figure 3). Imaging Studies. We performed imaging studies on all three radiotracers in the BALB/c nude mice bearing the U87MG human glioma xenografts. Figure 6 illustrates representative SPECT images of tumor-bearing mice administered with ∼400 µCi of [99mTc(SQ168)(EDDA)] (left), [99mTc(SQ168)(tricine)(TPPTS)] (middle), and [99mTc(SQ168)(tricine)(PDA)] (right). In general, tumors in all three SPECT images are clearly visualized at 4 h postinjection. The complex [99mTc(SQ168)(tricine)(TPPTS)] has the least activity accumulation in the abdominal region while [99mTc(SQ168)(EDDA)] has relatively low kidney uptake (Figure 6), which is completely consistent with the ex vivo biodistribution data at the same time point (Figure 3). Among three radiotracers evaluated in this study, [99mTc(SQ168)(tricine)(TPPTS)] has the best imaging quality at 4 h postinjection due to its rapid clearance from liver and lungs in BALB/c nude mice bearing the U87 human glioma xenografts. Metabolic Properties. We performed metabolism studies on all three radiotracers using normal BALB/c nude mice. Since they are excreted from both renal and hepatobiliary routes, we analyzed the samples from both liver and urine to determine if these radiotracers retain their chemical integrity at 2 h postinjection. Figure 7 illustrates the radio-HPLC chromatograms of [99mTc(SQ168)(tricine)(PDA)] (left) and [99mTc(SQ168)(tricine)(TPPTS)] (right) in the kit matrix before injection (A), in the liver (B), and in the urine (C). It is quite clear that [99mTc(SQ168)(tricine)(TPPTS)] retained its integrity while [99mTc(SQ168)(tricine)(PDA)] showed a certain degree of metabolism (10-15%) in both urine and liver samples at 2 h postinjection.

Figure 5. Direct comparison of excretion kinetics between [99mTc(SQ168)(EDDA)], [99mTc(SQ168)(tricine)(PDA)], and [99mTc(SQ168)(tricine)(TPPTS)] in BALB/c nude mice bearing the U87MG human glioma xenografts.

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Figure 6. SPECT images of BALB/c nude mice bearing the U87MG human glioma xenografts administered with ∼400 µCi of [99mTc(SQ168)(EDDA)] (left), [99mTc(SQ168)(tricine)(TPPTS)] (middle) and [99mTc(SQ168)(tricine)(PDA)] (right) at 4 h postinjection. Arrows indicate the location of glioma tumors.

Figure 7. Radio-HPLC chromatograms of [99mTc(SQ168)(tricine)(PDA)] (left) and [99mTc(SQ168)(tricine)(TPPTS)] (right) in the kit matrix before injection (A), in the liver (B), and in the urine (C) at 2 h postinjection.

The metabolic patterns of [99mTc(SQ168)(EDDA)] are very similar to those of [99mTc(SQ168)(tricine)(PDA)] (Figure SI).

DISCUSSION It is well established that the integrin Rvβ3 overexpressed on the neovasculature of many solid tumors, including neuroblastomas, glioblastomas, melanomas, lung carcinomas, and breast, prostate, and bladder cancers (6, 40-50). The integrin Rvβ3 expression level also correlates well with tumor progression in melanoma, glioma, and ovarian and breast cancers (7, 40-47). For the past decade, many cyclic RGD peptides and nonpeptide RGD mimetics have been labeled with different radioisotopes to develop the angiogenesis-targeting radiotracers for both diagnosis (125I, 99mTc, 111In, 18F, and 64Cu) and tumor-specific radiotherapy (90Y and 177Lu) (17-35). Since integrin Rvβ3 is overexpressed on the surface of the “activated” endothelial cells

of tumor neovasculature, the integrin Rvβ3-targeted radiotracers would be specific for rapidly growing and metastatic tumors. In general, the radiolabeled RGD peptide radiotracer of clinical importance should have high affinity and specificity for integrin Rvβ3. In the last several years, we and others have been using the “multivalency” concept to improve Rvβ3 binding affinity and the tumor targeting of the radiolabeled RGD peptides (11, 13, 29-35, 51-55). It is found that the use of a multimeric cyclic RGD peptide could significantly increase the affinity of radiotracers for integrin Rvβ3 through either simultaneous receptor binding or reduction of dissociation from the integrin Rvβ3 binding sites. For example, the integrin Rvβ3 binding affinity of E[c(RGDfK)]2 (a cyclic RGDfK dimer) is 10-fold higher than that of its monomeric analogue c(RGDfK) (33). In athymic female BALB/c nude mice with subcutaneous OVCAR-3 ovarian cancer xenografts, the 99mTc-labeled RGDfK

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dimer had significantly higher tumor retention than its monomeric counterpart (33). Similar results were also obtained with 64Cu- and 90Y-labeled cyclic RGD dimer conjugates: DOTA-E[c(RGDfK)]2 and DOTA-E[c(RGDyK)]2 (27, 32, 34, 35). For the radiolabeled RGD peptide radiotracer to be clinically useful, it is necessary to demonstrate its capability to image the integrin Rvβ3 expression in animal models bearing solid tumors of different origin. Janssen and co-workers evaluated the biodistribution characteristics of [99mTc(SQ168)(tricine)(TPPTS)] in BALB/c nude mice with subcutaneous OVCAR-3 ovarian cancer xenografts (33). Recently, we reported the evaluation of [99mTc(SQ168)(tricine)(L)] (L ) TPPTS, ISONIC, and PDA) in nude mice bearing the MDA-MB-435 human breast cancer xenografts (31). In this study, we use the BALB/c nude mice bearing the U87MG human glioma xenografts to examine biodistribution properties of three radiotracers, [99mTc(SQ168)(EDDA)], [99mTc(SQ168)(tricine)(PDA)], and [99mTc(SQ168)(tricine)(TPPTS)], and their capability to image the glioma integrin Rvβ3 expression. On the basis of biodistribution data, it is clear that all three radiotracers are able to localize in glioma tumors with moderately high tumor uptake (>2% ID/g over 4 h study period) and long tumor retention time. Results from the blocking experiment suggest that their tumor localization is integrin Rvβ3-mediated. SPECT imaging studies have demonstrated that the glioma tumors can be clearly visualized with all three radiotracers at 4 h postinjection. On the basis of their clearance patterns from blood, muscle, liver, and lungs, it seems that [99mTc(SQ168)(tricine)(TPPTS)] has the best imaging quality (Figure 6). Several approaches have been used to modify pharmacokinetics of the radiolabeled cyclic RGD peptides. For example, a sugar moiety has been used to improve the hepatobiliary clearance of 18F-labeled cyclic RGD peptides (17-19). The poly(ethylene glycol) linking groups are also useful for improving both tumor uptake and liver clearance of radiolabeled cyclic RGD peptides (23, 24, 27, 52-54). A number of coligands have been used to modify biological properties of the 99mTc-labeled HYNIC-biomolecule conjugates (29, 31, 36-38, 56-60). For example, it was found that the coligands had significant impact on biodistribution properties of complexes [99mTc(fMLFKHYNIC)(L)2] (L ) glucoheptonate, mannitol, glucamine, and tricine) (57). [99mTc(HYNIC-TOC)(tricine)2] (TOC ) Tyr3octreotide) was found to have higher protein binding than [99mTc(HYNIC-TOC)(EDDA)] and [99mTc(HYNIC-TOC)(tricine)(NA)] (58, 59). It was reported that the best coligand combinations are tricine/NIC and tricine/ISONIC for the 99mTclabled HYNIC-CCK8 (CCK8 ) cholecystokinin peptides) conjugates (60). In our previous study, we also found that [99mTc(SQ168)(tricine)(PDA)] had lower liver uptake in the athymic nude mice bearing the MDA-MB-435 human breast cancer xenografts over 2 h study period, and better tumor/liver ratios than [99mTc(SQ168)(tricine)(TPPTS)] at >60 min postinjection (31). In this study, [99mTc(SQ168)(tricine)(TPPTS)] was found to have the lowest liver uptake (Figure 5) and the best imaging quality (Figure 6) in BALB/c nude mice bearing U87MG human glioma xenografts. It also has the highest metabolic stability (Figure 7) in normal BALB/c nude mice. We believe that this discrepancy is most likely caused by the animal species. Therefore, caution must be taken when comparing biodistribution and excretion characteristics of radiotracers in different animals.

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The choice of coligand for routine preparation of complexes [99mTc(SQ168)(tricine)(L)] (L ) TPPTS, NIC, ISONIC, and PDA) depends on several factors. For example, the radiotracer must demonstrate its capability to image the tumor integrin Rvβ3 expression in various animal models. It must be excreted via the renal route to minimize the unwanted radioactivity accumulation in the abdominal region. It should have a high metabolic stability. In addition, a kit formulation is needed for routine preparation of the radiotracer. From the formulation development point of view, TPPTS has the obvious advantage over ISONIC and PDA since it is a strong reducing agent, and stannous chloride is not needed for successful preparation of [99mTc(SQ168)(tricine)(TPPTS)]. A non-SnCl2 formulation has been developed for routine 99mTc-labeling of the HYNICconjugated cyclic RGD peptides (61). Since TPPTS is able to “extract” the oxygen from [99mTc]pertechnetate, there is virtually no [99mTc]colloid formation if the non-SnCl2 formulation is used for 99mTc-labeling. On the basis of the results from this and our previous studies, it is concluded that coligands in ternary ligand 99mTc complexes [99mTc(SQ168)(tricine)(L)] (L ) TPPTS, NIC, ISONIC, and PDA) have minimal effect on their tumor-targeting capability. The coligand effect on excretion kinetics and metabolic fate of the radiotracer is not completely consistent in two animal species. Therefore, their biodistribution patterns and metabolic stability must be verified in larger rodents or mammals, which are more indicative of those in humans.

CONCLUSION The key finding of this study is that all three radiotracers, [99mTc(SQ168)(EDDA)], [99mTc(SQ168)(tricine)(PDA)], and [99mTc(SQ168)(tricine)(TPPTS)], are able to localize in glioma tumors via integrin Rvβ3-binding. The coligand has minimal effect on the integrin Rvβ3-targeting capability of radiotracers, but it has a significant impact on their biodistribution properties. Results from the SPECT imaging studies show that glioma tumors can be clearly visualized with all three radiotracers at 4 h postinjection. On the basis of their clearance patterns from blood, muscle, liver, and lungs, it seems that [99mTc(SQ168)(tricine)(TPPTS)] has the best imaging quality and is a promising candidate for more preclinical evaluations in the future.

ACKNOWLEDGMENT Authors would thank Professor Jingde Zhu, Shanghai Cancer Research Institute, for providing the U87MG human glioma cells. This work is supported, in part, by Peking University HealthScienceCenter(F.W.)andresearchgrants: Z00004105040311 (F.W.) from Beijing Science and Technology Program and BCTR0503947 (S.L.) from Susan G. Komen Breast Cancer Foundation. Supporting Information Available: Biodistribution data (%ID/ g) for [99mTc(SQ168)(EDDA)], [99mTc(SQ168)(tricine)(PDA)], and [99mTc(SQ168)(tricine)(TPPTS)] are listed in Tables S1S3. Figure SI shows the radio-HPLC chromatograms of [99mTc(SQ168)(EDDA)] in kit matrix, urine, and feces at 2 h postinjection. These materials are available free of charge at http://pubs.acs.org.

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