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Basic Science Investigation |
vß3 Integrin Expression
Molecular Imaging Program at Stanford (MIPS) and Bio-X Program, Department of Radiology, Stanford University School of Medicine, Stanford, California
Correspondence: For correspondence or reprints contact: Xiaoyuan Chen, PhD, Molecular Imaging Program at Stanford (MIPS) and Bio-X Program, Department of Radiology, Stanford University School of Medicine, 1201 Welch Rd., Room P095, Stanford, CA 94305-5484. E-mail: shawchen{at}stanford.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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vß3 plays a key role in tumor angiogenesis and metastasis. A series of 18F-labeled RGD peptides have been developed for PET of integrin expression based on primary amine-reactive prosthetic groups. In this study we introduced a new method of labeling RGD peptides through a thiol-reactive synthon, N-[2-(4-18F-fluorobenzamido)ethyl]maleimide (18F-FBEM). Methods: 18F-FBEM was synthesized by coupling N-succinimidyl 4-18F-fluorobenzoate (18F-SFB) with N-(2-aminoethyl)maleimide. After high-pressure liquid chromatography purification, it was allowed to react with thiolated RGD peptides, and the resulting tracers were subjected to receptor-binding assay, in vivo metabolic stability assessment, biodistribution, and microPET studies in murine xenograft models. Results: Conjugation of monomeric and dimeric sulfhydryl-RGD peptides with 18F-FBEM was achieved in high yields (85% ± 5% nondecay-corrected on the basis of 18F-FBEM). The radiochemical purity of the 18F-labeled peptides was >98% and the specific activity was 100
150 TBq/mmol. Noninvasive microPET and direct tissue sampling experiments demonstrated that both 18F-FBEM-SRGD (RGD monomer) and 18F-FBEM-SRGD2 (RGD dimer) had integrin-specific tumor uptake in subcutaneous U87MG glioma and orthotopic MDA-MB-435 breast cancer xenografts. Conclusion: The new tracer 18F-FBEM-SRGD2 was synthesized with high specific activity via 18F-FBEM and the tracer exhibited high receptor-binding affinity, tumor-targeting efficacy, metabolic stability, as well as favorable in vivo pharmacokinetics. The new synthon 18F-FBEM developed in this study will also be useful for radiolabeling of other thiolated biomolecules.
Key Words: thiol-reactive synthon • 18F-FBEM • microPET • 18F labeling • integrin vß3
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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vß3 integrin, which binds to arginine-glycine-aspartic acid (RGD)-containing components of the interstitial matrix, such as vitronectin, fibronectin, and thrombospondin, is significantly upregulated on endothelium during angiogenesis but not in quiescent endothelium (1,2). The special role of integrin vß3 in tumor invasion and metastasis arises from its ability to recruit and activate matrix metalloproteinase MMP-2 and plasmin, which degrade components of the basement membrane and interstitial matrix (3). Integrins expressed on endothelial cells modulate cell migration and survival during angiogenesis, whereas integrins expressed on carcinoma cells potentiate metastasis by facilitating invasion and movement across blood vessels (2,4). Antagonists of integrin vß3 (antibodies, peptides, and peptidomimetics) can inhibit tumor angiogenesis, tumor growth, and metastasis in vivo (5). The ability to noninvasively visualize and quantify tumor integrin vß3 expression level will provide new opportunities to document tumor (tumor cells and sprouting tumor vasculature) integrin expression, to more appropriately select patients for antiintegrin treatment, and to monitor treatment efficacy in integrin-positive patients.
Over the last several years, significant advances have been achieved in developing novel probes for multimodality molecular imaging of tumor integrin expression (6). Small molecules, peptides, peptidomimetic integrin vß3 antagonists, and antibodies have been labeled with radioisotopes, superparamagnetic nanoparticles, fluorescent dyes, quantum dots, and microbubbles for PET, SPECT, MRI, near-infrared fluorescence, and ultrasound imaging of small animals, mostly tumor models (6,7). Because of the high sensitivity and adequate spatial and temporal resolution of PET, development of PET probes for integrin expression imaging is currently the most active among all of these modalities.
Cyclic RGD peptide was first labeled with 18F by Haubner et al. and the resulting 18F-galacto-RGD exhibited integrin vß3–specific tumor uptake in an integrin-positive M21 melanoma xenograft model (8,9). Initial clinical trials in healthy volunteers and a limited number of cancer patients revealed that this tracer can be administered safely to patients and is capable of delineating certain lesions that are integrin positive with some indication of integrin vß3 expression level in vivo (9,10). We labeled a series of mono-, di-, and tetrameric RGD peptides with 18F or 64Cu for integrin-positive tumor targeting (11–19). In particular, the dimeric RGD peptide-based tracer 18F-FRGD2 was found to be able to visualize and quantify the integrin expression level in vivo (11,12).
Radiofluorination of RGD peptides generally uses 18F-synthons such as N-succinimidyl 4-18F-fluorobenzoate (18F-SFB) (20,21) or p-nitrophenyl 18F-fluoropropionate (18F-NFP) (22) to form a stable amide bond by reacting with primary amino groups of RGD peptides. It was also reported that oxoamino derivatives of RGD peptides react with 4-18F-fluorobenzaldehyde (18F-FBA) under acidic condition to form an oxime (23). A few 18F labeled thiol-reactive reagents have been reported in the literature—namely, 1-(4-18F-fluorophenyl)pyrrole-2,5-dione (18F-FPPD) (24), N-[3-(2,5-dioxo-2,5-dihydropyrrol-1-yl)phenyl]-4-18F-fluorobenzamide (18F-DDPFB) (24), 1-[3-(2-(18F-fluoropyridin-3-yloxy)propyl]pyrrole-2,5-dione (18F-FPyME) (25), and N-[4-[(4-18F-fluorobenzylidene)aminooxyl]butyl]maleimide (18F-FBABM) (26). However, no in vivo microPET data have been reported on tracers synthesized using these prosthetic groups. In this study, we developed a new thiol-reactive synthon, N-[2-(4-18F-fluorobenzamido)ethyl]maleimide (18F-FBEM), for 18F labeling of thiol-containing molecules. Two thiolated RGD peptides were labeled with 18F through 18F-FBEM and tested in murine xenograft models.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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The SATA-RGD peptides were prepared following the protocol supplied by the vender. Briefly, c(RGDyK) or E[c(RGDyK)]2 (5 µmol) in 1 mL 50 mmol/L Na2B4O7 buffer (pH 8.5) was mixed with 100 µL SATA solution in dimethyl sulfoxide (DMSO; 6 µmol). After the reaction had gone to completion as shown by analytic RP-HPLC, it was quenched by 100 µL 2% trifluoroacetic acid (TFA) in water. The crude product was lyophilized without purification. The yield of SATA-c(RGDyK) (HPLC retention time [Rt], 12.1 min) was 95% and that of SATA-E[c(RGDyK)]2 (Rt, 13.6 min) was 65% on the basis of analytic RP-HPLC.
The crude SATA-RGD peptides (20 mg) were dissolved in 1 mL water and 100 µL of 0.5 mol/L hydroxylamine solution were added. The pH was adjusted to 6.0. After 2 h, sulfhydryl-c(RGDyK) and sulfhydryl-E[c(RGDyK)]2 (denoted as SRGD and SRGD2, respectively) were purified by semipreparative RP-HPLC. The overall yield was 80% and 50% for SRGD (Rt, 10.7 min) and SRGD2 (Rt, 13.1 min), respectively; little or no dimerization was observed for either peptide when stored under acidic condition (pH 3). MALDI-TOF MS (matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy): SRGD, C29H43N9O9S, calculated 693.3, observed 694.5 ([M+H]+); SRGD2, C61H89N19O19S, calculated 1,423.6, observed 1,422.7 ([M+H]+).
N-(2-Aminoethyl)maleimide (5 µmol) in 200 µL acetonitrile, N-succinimidyl 4-fluorobenzoate (4.5 µmol) in 100 µL acetonitrile, and 500 µL 50 mmol/L Na2B4O7 buffer (pH 8.5) were mixed and reacted at 50°C for 20 min. The reaction was quenched by adding 100 µL 2% TFA in water. HPLC purification gave FBEM in 85% yield. 1H NMR (chloroform-d, 400 MHz): 7.79–7.76 (m, 2H, phenyl o-H); 7.26–7.09 (m, 2H, phenyl m-H); 6.75 (2H, CH=CH); 3.86–3.65 (m, 4H, CH2–CH2).
The sulfhydryl-RGD peptides (0.5 µmol) were dissolved in 0.5 mL phosphate-buffered saline (PBS, pH 7.4; Invitrogen Corp.). FBEM (0.55 µmol) was dissolved in 200 µL acetonitrile and added to the solution. After 30 min, the reaction mixture was subjected to HPLC purification. FBEM-SRGD (Rt, 14.8 min) and FBEM-SRGD2 (Rt, 15.2 min) were obtained with 80% yield. MALDI-TOF MS: FBEM-SRGD, C42H54FN11O12S, calculated 956.1, observed 956.7 ([M+H]+); FBEM-SRGD2, C73H98FN21O22S, calculated 1,672.5, observed 1,673.5 ([M+H]+).
Radiochemistry
18F-SFB was synthesized as previously reported (12,15) with C18 Sep-Pak cartridge purification. It was dissolved in acetonitrile (300 µL) and 1 mg N-(2-aminoethyl)maleimide in 500 µL acetonitrile and 20 µL DIPEA were added. The reaction mixture was heated to 40°C for 20 min and then quenched by addition of 50 µL TFA. HPLC purification gave 18F-FBEM (Rt, 13.9 min; total reaction time, 150 ± 20 min with nondecay-corrected radiochemical yield of 5% ± 2% on the basis of 18F-F–; specific activity, 150200 TBq/mmol).
18F-FBEM was dissolved in 600 µL PBS buffer (pH 7.4); 0.2 mg of sulfhydryl-RGD peptide in 50 µL DMSO and 1.0 mg TCEP·HCl in 0.1 mL water were then added. The pH was adjusted to 7.07.5 using 0.2 mol/L NaOH solution. The reaction mixture was kept at room temperature (r.t.) for 20 min. HPLC purification gave 18F-FBEM-SRGD (Rt, 14.9 min) and 18F-FBEM-SRGD2 (Rt, 15.3 min) in 80% nondecay-corrected yield in both cases. The radiotracers were reconstituted in PBS and passed through a 0.22-µm Millipore filter into a sterile vial for in vivo applications. Nonreacted sulfhydryl-RGD peptides were baseline-separated from the desired products during HPLC and the specific activity of the tracers was 100–150 TBq/mmol.
Eppendorf microcentrifuge tubes containing 500 µL of octanol, 500 µL of normal saline, and 370 kBq of 18F-FBEM-SRGD or 18F-FBEM-SRGD2 were vortexed vigorously for 1 min. Each tube was centrifuged at 14,000 rpm for 5 min and the activities in 20-µL aliquots of both organic and aqueous layers were measured by a 
-counter (GMI, Inc.). The reported octanol/water partition coefficient represents the mean ± SD of 6 measurements.
In Vitro Cell-Binding Assay
Both U87MG and MDA-MB-435 cell lines were purchased from American Type Culture Collection and the culture media were obtained from Invitrogen Co. U87MG glioblastoma cells were grown in Dulbecco's modified Eagle medium (DMEM, low glucose) and MDA-MB-435 breast cancer carcinoma cells were grown in Leibovitz's L-15 medium. Both cell lines were cultured in the medium supplemented with 10% (v/v) fetal bovine serum (FBS) at 37°C. In vitro integrin-binding affinities and specificities were assessed via displacement cell-binding assays using 125I-echistatin as the integrin vß3–specific radioligand. Both U87MG and MDA-MB-435 cells are integrin vß3 positive (12). Cell-binding assay were performed using U87MG cells. The cells were harvested, washed twice with PBS, and resuspended (2 x 106 cells/mL) in binding buffer (20 mmol/L Tris, pH 7.4, 150 mmol/L NaCl, 2 mmol/L CaCl2, 1 mmol/L MgCl2, 1 mmol/L MnCl2, 0.1% bovine serum albumin). Millipore 96-well filter multiscreen DV plates (pore size, 0.65 µm) were seeded with 105 cells per well and incubated with 125I-echistatin (30,000 cpm/well) in the presence of increasing concentrations of different RGD peptide analogs (0–1,000 nmol/L). The total volume in each well was adjusted to 200 µL. After incubation at r.t. for 2 h, the plates were filtered through a multiscreen vacuum manifold and washed twice with cold binding buffer. The filters were collected and the radioactivity was measured using a -counter. The best-fit IC50 (inhibitory concentration of 50%) values for U87MG cells were calculated by fitting the data by nonlinear regression using GraphPad Prism (GraphPad Software, Inc.). Experiments were performed twice with triplicate samples.
Animal Models
All animal experiments were performed under a protocol approved by the Stanford University Administrative Panel on Laboratory Animal Care. The MDA-MB-435 breast cancer model was established by orthotopic injection of 5 x 106 cells into the left mammary fat pad of female athymic nude mice, whereas the U87MG tumor model was obtained by injecting a mixture of 5 x 106 cells suspended in 50 µL medium and 50 µL Matrigel (BD Biosciences) into the right front leg. The mice were used for biodistribution and microPET imaging studies when the tumor volume reached 300–400 mm3 (3–4 wk after inoculation for both U87MG and MDA-MB-435 tumors).
Biodistribution Studies
Female athymic nude mice bearing both U87MG and MDA-MB-435 tumors were injected with 1 MBq of 18F-FBEM-SRGD or 18F-FBEM-SRGD2. The mice were sacrificed and dissected at 10, 30, and 60 min after injection. Blocking experiment was performed by coinjecting radiotracer with a saturating dose of c(RGDyK) (10 mg/kg mouse body weight) and the mice were sacrificed at 60 min after injection. Blood, tumor, major organs, and tissues were collected and wet weighed (contents in the intestines were removed before weighing). The radioactivity in the tissues was measured using a -counter. The results were calculated as percentage injected dose per gram (%ID/g). For each mouse, the radioactivity of the tissue samples was calibrated against a known aliquot of the injectate and normalized to a body weight of 20 g. Values are expressed as mean ± SD for 3 animals per group.
microPET and Image Analysis
PET scans were performed using a microPET R4 rodent model scanner (Concorde Microsystems Inc.). The scanner has a computer-controlled bed and 10.8-cm transaxial and 8-cm axial fields of view (FOVs). It has no septa and operates exclusively in the 3-dimensional list mode. Animals were placed near the center of FOV of the microPET scanner, where the highest image resolution and sensitivity are available. Mice were injected with 3.7 MBq of 18F-FBEM-SRGD or 18F-FBEM-SRGD2 via tail vein under isoflurane anesthesia. The 60-min dynamic (5 x 60 s, 5 x 120 s, 5 x 180 s, 6 x 300 s) microPET data acquisition (total of 21 frames) was started about 3 min after radiotracer injection. Later time-point static images were also acquired as 10-min static images after obtaining a 1-h dynamic scan. The images were reconstructed by a 2-dimensional ordered-subsets expectation maximum algorithm and no correction was applied for attenuation or scatter (27).
For each microPET scan, regions of interests (ROIs) were drawn over each tumor, normal tissue, and major organs by using vendor software ASI Pro 5.2.4.0 on decay-corrected whole-body coronal images. The maximum radioactivity concentration (accumulation) within a tumor or an organ was obtained from mean pixel values within the multiple ROI volume, which were converted to MBq/mL/min by using a conversion factor. Assuming a tissue density of 1 g/mL, the ROIs were converted to MBq/g/min, and then divided by the administered activity to obtain an imaging ROI-derived %ID/g.
Metabolic Stability
Athymic nude mice bearing U87MG tumor were intravenously injected 3.7 MBq of 18F-FBEM-SRGD2. The animals were sacrificed 60 min after tracer injection. Blood, urine, liver, kidneys, and tumor were collected. Blood was immediately centrifuged for 5 min at 13,200 rpm. Organs were homogenized using an IKA Ultra-Turrax T8 homogenizer (IKA Works Inc.), suspended in 1 mL of PBS buffer, and centrifuged for 5 min at 13,200 rpm. After removal of the supernatant, the pellets were washed with 500 µL of PBS. For each sample, supernatants of both centrifugation steps were combined and passed through Sep-Pak C18 cartridges. The urine sample was directly diluted with 1 mL of PBS and then passed through the cartridge. The cartridges were washed with 2 mL of water and eluted with 2 mL of acetonitrile containing 0.1% TFA. After removal of acetonitrile, the residue was redissolved in 1 mL of water and injected onto an analytic HPLC column. Radioactivity was monitored using a solid-state radiation detector. The eluent was also collected using a fraction collector (0.5 min/fraction) and the activity of each fraction was measured by a -counter.
Statistical Analysis
Quantitative data are expressed as mean ± SD. Means were compared using 1-way ANOVA and a Student t test. P values < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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18F-SFB was synthesized following a previously reported procedure (12,15). 18F-FBEM was obtained by coupling 18F-SFB with N-(2-aminoethyl)maleimide (Fig. 1A). After HPLC purification, the thiol-reactive synthon was allowed to react with SRGD or SRGD2 at r.t. for 20 min. Starting from 18F-F–, the total reaction time including final HPLC purification was about 200 ± 25 min. The overall decay-corrected radiochemical yield was 20% ± 4% (n = 5) for both 18F-FBEM-SRGD (Fig. 1B) and 18F-FBEM-SRGD2 (Fig. 1C). On the basis of 18F-FBEM, both reactions were achieved in high yields (85% ± 5% nondecay corrected), virtually quantitative. The radiochemical purity of the 18F-labeled peptides was >98% according to analytic HPLC. The octanol/water partition coefficient (log P) for 18F-FBEM-SRGD was determined to be 0.93 ± 0.02, indicating the hydrophobic character of this tracer, whereas 18F-FBEM-SRGD2 was hydrophilic (log P = –1.69 ± 0.02).
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vß3–specific binding of the radiotracer 18F-FBEM-SRGD2.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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Thiol-reactive agents have been used to modify peptides and proteins at specific sites, providing high chemoselectivity as compared with amine and carboxylate-reactive reagents (32,33). The disulfide bonds of a protein can be reduced to enable modification using thiol-specific reagents (34–36). More recently, site-directed mutagenesis was used to place cysteine residues on the surface of proteins to provide reactive sulfhydryl groups (37,38). Several thiol-reactive 18F-synthons have been described (24–26), all of which bear a maleimide group allowing for thiol-specific Michael addition reaction. The total synthesis time (100–150 min) and radiochemical yield (10%–20% nondecay corrected) of these synthons are comparable to 18F-FBEM. However, no in vivo data have been reported on tracers synthesized using these prosthetic groups.
We have labeled c(RGDyK) with 18F using 18F-SFB as a prosthetic group (18). The labeling yield was reasonably good for in vivo imaging applications. The resulting 18F-FB-RGD had good tumor-to-blood and tumor-to-muscle ratios but also rapid tumor washout and unfavorable hepatobiliary excretion, making it suitable only for visualizing lesions above the liver (e.g., breast cancer, head and neck cancer, and brain tumor). Because the bent conformation of c(RGDyK) has been optimized to fit into the deep cleft between the - and ß-units of integrin vß3, it is unlikely that one can further improve integrin affinity and selectivity of the monomeric RGD peptide by fine tuning the pentapeptide configuration (39). Therefore, the polyvalency effect has been applied to develop dimeric and multimeric RGD peptides, with repeating cyclic pentapeptide units connected by glutamates (11,12,14,19,40). We have found that 18F-FRGD2 (18F-FB-E[c(RGDyK)]2) had predominant renal excretion and almost twice as much tumor uptake in the same animal model as compared with the monomeric tracer 18F-FB-RGD (11,12). The synergistic effect of polyvalency and improved pharmacokinetics may be responsible for the excellent imaging characteristics of 18F-FRGD2. At late time points when most of the nonspecific binding had been cleared, the tumor-to-background ratio also had a linear relationship with the tumor integrin levels, thus making it possible to quantify the tumor integrin expression level in vivo with static PET scans using 18F-FRGD2. We are currently in the process of translating 18F-FRGD2 into clinical trials. In parallel, we developed 18F-FBEM as a prosthetic group for dimeric RGD peptide labeling as well as for protein or engineered antibody labeling through site-specific Michael addition with the sulfhydryl group. The reaction between 18F-FBEM and the thiolated RGD peptides was virtually quantitative. Although 18F-FBEM-SRGD2 demonstrated integrin specificity, as evidenced by effective inhibition of tumor activity accumulation in the presence of a blocking dose of integrin vß3 antagonist c(RGDyK), whether it is able to quantify integrin expression in vivo remains to be determined in future studies. It is also worth noting that addition of the thiolated RGD peptides to 18F-FBEM generates a new asymmetric center, resulting in 2 diastereomeric products. Because we only observed one sharp peak in the analytic HPLC for both 18F-FBEM-SRGD and 18F-FBEM-SRGD2, it is very likely that such a small change was not detectable by the HPLC system used.
This study demonstrated that 18F-FBEM could be used to efficiently label peptides through the sulfhydryl group. The major advantage of 18F-FBEM lies in the fact that it can be applied to label a variety of peptides, proteins, or oligonucleotides containing sulfhydryl groups. Because most proteins contain Cys residues, whereas those that do not can be easily engineered to incorporate a Cys residue without compromising the biologic activity, it is expected that 18F-FBEM will have wide applications in the near future for development of novel tracers for in vivo PET.
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CONCLUSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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vß3 expression level in vivo). 18F-FBEM also provides a general method of labeling thiol-containing peptides, proteins, antibodies, as well as 5'-thio-functionalized oligonucleotides in high radiochemical yield and high specific activity for successful PET applications. |
ACKNOWLEDGMENTS |
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FOOTNOTES |
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COPYRIGHT © 2006 by the Society of Nuclear Medicine, Inc.
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References |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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vß3. Cell. 1996;85:683–693.[Medline]vß3 as a therapeutic target for blocking tumor-induced angiogenesis. Curr Drug Targets. 2003;4:123–131.[Medline]vß3. Biotechniques. 2005;39(suppl):S6–S17.vß3 integrin expression using 18F-labeled RGD-containing glycopeptide and positron emission tomography. Cancer Res. 2001;61:1781–1785.vß3 integrin in cancer patients by positron emission tomography and [18F]galacto-RGD. PLoS Med. [serial online]. 2005;2:e70.[Medline]vß3-selective tracer 18F-galacto-RGD in cancer patients. J Nucl Med. 2005;46:1333–1341.vß3-integrin expression with 18F-labeled dimeric RGD peptide. Mol Imaging. 2004;3:96–104.[Medline]vß3 expression with [18F]FRGD2. J Nucl Med. 2006;47:113–121.vß3-integrin expression. J Nucl Med. 2004;45:1776–1783.v-integrin expression with 64Cu-labeled dimeric RGD peptides. Mol Imaging Biol. 2004;6:350–359.[Medline]vß3-targeted imaging of lung cancer. Neoplasia. 2005;7:271–279.[Medline]v-integrin expression using 18F- and 64Cu-labeled RGD peptide. Bioconjug Chem. 2004;15:41–49.[Medline]v-integrin expression using 64Cu-labeled tetrameric RGD peptide. J Nucl Med. 2005;46:1707–1718.vß3 in complex with an Arg-Gly-Asp ligand. Science. 2002;296:151–155.Related articles in JNM:
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