Dimeric RGD Research Articles

177Lu‐labeled monomeric, dimeric and multimeric RGD peptides for the therapy of tumors expressing α(ν)β(3) integrins

The conjugation of peptides to gold nanoparticles (AuNPs) produces biocompatible and stable multimeric systems with target‐specific molecular recognition. Peptides based on the cyclic Arg‐Gly‐Asp (RGD) sequence have been reported as high‐affinity agents for the α(ν)β(3) integrin. The aim of this research was to prepare a multimeric system of 177Lu‐labeled gold nanoparticles conjugated to c(RGDfK)C (cyclo(Arg‐Gly‐Asp‐Phe‐Lys)Cys) and to compare the radiation‐absorbed dose with that of 177Lu‐labeled monomeric and dimeric RGD peptides to α(ν)β(3) integrin‐positive U87MG tumors in mice. DOTA‐GGC (1,4,7,10‐tetraazacyclododecane‐N‐N′,N″,N‴‐tetraacetic acid‐Gly‐Gly‐Cys) and c(RGDfK)C peptides were synthesized and conjugated to AuNPs by a spontaneous reaction of the thiol groups. Transmission electron microscopy, ultraviolet–visible, X‐ray photoelectron spectroscopy, Raman and far‐infrared spectroscopy techniques demonstrated that AuNPs were functionalized with the peptides. For the 177Lu‐AuNP‐c(RGDfK)C to be obtained, the 177Lu‐DOTA‐GGC radiopeptide was first prepared and added to a solution of AuNPs followed by c(RGDfK)C (25 µl, 5 µ m) at 18 °C for 15 min. 177Lu‐DOTA‐GGC, 177Lu‐DOTA‐cRGDfK and 177Lu‐DOTA‐E‐c(RGDfK)2 were prepared by adding 177LuCl3 (370 MBq) to 5 µl (1 mg/ml) of the DOTA derivative diluted with 50 µl of 1 m acetate buffer pH 5. The mixture was incubated at 90 °C in a block heater for 30 min. Radiochemical purity was determined by ultrafiltration and HPLC analyses. Biokinetic studies were accomplished in athymic mice with U87MG‐induced tumors. The radiochemical purity for all 177Lu‐RGD derivatives was 96 ± 2%. 177Lu‐absorbed doses per injected activity delivered to U87MG tumors were 0.357 ± 0.052 Gy/MBq (multimer), 0.252 ± 0.027 Gy/MBq (dimer) and 0.102 ± 0.018 Gy/MBq (monomer). 177Lu‐labeled dimeric and multimeric RGD peptides demonstrated properties suitable for targeted radionuclide therapy of tumors expressing α(ν)β(3) integrins. Copyright © 2012 John Wiley & Sons, Ltd.

Design, Preparation and Characterization of Cyclic RGD Dimer for Targeting Integrin aVb3

Design, Preparation and Characterization of Cyclic RGD Dimer for Targeting Integrin aVb3

Science to Practice: Pilot Study of FPPRGD2 for Imaging αvβ3Integrin—How Integral Are Integrins?

Mittra et al (1) describe a new positron emission tomographic (PET) radiopharmaceutical, fluorine 18 ((18)F) 2-fl uoropropionyl labeled PEGylated dimeric RGD peptide (FPPRGD2), a marker of α(v)β(3) integrin expression, which they tested in fi ve healthy volunteers. No adverse events were encountered, and the biodistribution suggested both a renal and a hepatobiliary excretory route. This early safety testing paves the way for future studies of patients with cancer who are undergoing antiangiogenic therapies. (18)F-FPPRGD2 represents one of several integrin imaging agents that are moving closer to clinical reality.

One-step radiosynthesis of 18F-AlF-NOTA-RGD2 for tumor angiogenesis PET imaging

One of the major obstacles of the clinical translation of (18)F-labeled arginine-glycine-aspartic acid (RGD) peptides has been the laborious multistep radiosynthesis. In order to facilitate the application of RGD-based positron emission tomography (PET) probes in the clinical setting we investigated in this study the feasibility of using the chelation reaction between Al(18)F and a macrocyclic chelator-conjugated dimeric RGD peptide as a simple one-step (18)F labeling strategy for development of a PET probe for tumor angiogenesis imaging. Dimeric cyclic peptide E[c(RGDyK)](2) (RGD(2)) was first conjugated with a macrocyclic chelator, 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), and the resulting bioconjugate NOTA-RGD(2) was then radiofluorinated via Al(18)F intermediate to synthesize (18)F-AlF-NOTA-RGD(2). Integrin binding affinities of the peptides were assessed by a U87MG cell-based receptor binding assay using (125)I-echistatin as the radioligand. The tumor targeting efficacy and in vivo profile of (18)F-AlF-NOTA-RGD(2) were further evaluated in a subcutaneous U87MG glioblastoma xenograft model by microPET and biodistribution. NOTA-RGD(2) was successfully (18)F-fluorinated with good yield within 40 min using the Al(18)F intermediate. The IC(50) of (19)F-AlF-NOTA-RGD(2) was determined to be 46 ± 4.4 nM. Quantitative microPET studies demonstrated that (18)F-AlF-NOTA-RGD(2) showed high tumor uptake, fast clearance from the body, and good tumor to normal organ ratios. NOTA-RGD(2) bioconjugate has been successfully prepared and labeled with Al(18)F in one single step of radiosynthesis. The favorable in vivo performance and the short radiosynthetic route of (18)F-AlF-NOTA-RGD(2) warrant further optimization of the probe and the radiofluorination strategy to accelerate the clinical translation of (18)F-labeled RGD peptides.

Pilot Pharmacokinetic and Dosimetric Studies of18F-FPPRGD2: A PET Radiopharmaceutical Agent for Imaging αvβ3Integrin Levels

To assess the safety, biodistribution, and dosimetric properties of the positron emission tomography (PET) radiopharmaceutical agent fluorine 18 ((18)F) FPPRGD2 (2-fluoropropionyl labeled PEGylated dimeric RGD peptide [PEG3-E{c(RGDyk)}2]), which is based on the dimeric arginine-glycine-aspartic acid (RGD) peptide sequence and targets α(v)β(3) integrin, in the first volunteers imaged with this tracer. The protocol was approved by the institutional review board, and written informed consent was obtained from all participants. Five healthy volunteers underwent whole-body combined PET-computed tomography 0.5, 1.0, 2.0, and 3.0 hours after tracer injection (mean dose, 9.5 mCi ± 3.4 [standard deviation] [351.5 MBq ± 125.8]; mean specific radioactivity, 1200 mCi/mmol ± 714 [44.4 GBq/mmol ± 26.4]). During this time, standard vital signs, electrocardiographic (ECG) readings, and blood sample values (for chemistry, hematologic, and liver function tests) were checked at regular intervals and 1 and 7 days after the injection. These data were used to evaluate tracer biodistribution and dosimetric properties, time-activity curves, and the stability of laboratory values. Significant changes in vital signs and laboratory values were evaluated by using a combination of population-averaged generalized estimating equation regression and exact paired Wilcoxon tests. The administration of (18)F-FPPRGD2 was well tolerated, with no marked effects on vital signs, ECG readings, or laboratory values. The tracer showed the same pattern of biodistribution in all volunteers: primary clearance through the kidneys (0.360 rem/mCi ± 0.185 [0.098 mSv/MBq ± 0.050]) and bladder (0.862 rem/mCi ± 0.436 [0.233 mSv/MBq ± 0.118], voiding model) and uptake in the spleen (0.250 rem/mCi ± 0.168 [0.068 mSv/MBq ± 0.046]) and large intestine (0.529 rem/mCi ± 0.236 [0.143 mSv/MBq ± 0.064]). The mean effective dose of (18)F-FPPRGD2 was 0.1462 rem/mCi ± 0.0669 (0.0396 mSv/MBq ± 0.0181). With an injected dose of 10 mCi (370 MBq) and a 1-hour voiding interval, a patient would be exposed to an effective radiation dose of 1.5 rem (15 mSv). Above the diaphragm, there was minimal uptake in the brain ventricles, salivary glands, and thyroid gland. Time-activity curves showed rapid clearance from the vasculature, with a mean 26% ± 17 of the tracer remaining in the circulation at 30 minutes and most of the activity occurring in the plasma relative to cells (mean whole blood-plasma ratio, 0.799 ± 0.096). (18)F-FPPRGD2 has desirable pharmacokinetic and biodistribution properties. The primary application is likely to be PET evaluation of oncologic patients-especially those with brain, breast, or lung cancer. Specific indications may include tumor staging, identifying patients who would benefit from antiangiogenesis therapy, and separating treatment responders from nonresponders early.

Rapid and Simple One-Step F-18 Labeling of Peptides

Labeling biomolecules with ¹⁸F is usually done through coupling with prosthetic groups, which requires several time-consuming radiosynthesis steps and therefore in low labeling yield. In this study, we designed a simple one-step ¹⁸F-labeling strategy to replace the conventional complex and the long process of multiple-step radiolabeling procedure. Both monomeric and dimeric cyclic RGD peptides were modified to contain 4-NO₂-3-CF₃ arene as precursors for direct ¹⁸F labeling. Binding of the two functionalized peptides to integrin α(v)β₃ was tested in vitro using the MDA-MB-435 human breast cell line. The most promising functionalized peptide, the dimeric cyclic RGD, was further evaluated in vivo in an orthotopic MDA-MB-435 tumor xenograft model. The use of relatively low amount of precursor (~0.5 μmol) gave reasonable yield, ranging from 7 to 23% (decay corrected, calculated from the start of synthesis) after HPLC purification. Overall reaction time was 40 min, and the specific activity of the labeled peptide was high. Modification of RGD peptides did not significantly change the biological binding affinities of the modified peptides. Small animal PET and biodistribution studies revealed integrin specific tumor uptake and favorable biokinetics. We have developed a novel one-step ¹⁸F radiolabeling strategy for peptides that contain a specific arene group, which shortens reaction time and labor significantly, requires low amount of precursor, and results in specific activity of 79 ± 13 GBq/μmol. Successful introduction of 4-fluoro-3-trifluoromethylbenzamide into RGD peptides may be a general strategy applicable to other biologically active peptides and proteins.

Two 90Y-Labeled Multimeric RGD Peptides RGD4 and 3PRGD2 for Integrin Targeted Radionuclide Therapy

We have recently developed a series of new Arg-Gly-Asp (RGD) dimeric peptides for specific targeting of integrin α(v)β₃ with enhanced tumor uptake and improved pharmacokinetics. In this study, we investigated ⁹⁰Y-labeled RGD tetramer (RGD4) and the new type of RGD dimer (3PRGD2), for the radionuclide therapy of integrin α(v)β₃-positive tumors. Biodistribution and gamma imaging studies of ¹¹¹In labeled RGD4 and 3PRGD2 were performed. Groups of nude mice were used to determine maximum tolerated dose (MTD) of ⁹⁰Y-DOTA-RGD4 and ⁹⁰Y-DOTA-3PRGD2. The radionuclide therapeutic efficacy of ⁹⁰Y-DOTA-RGD4 and ⁹⁰Y-DOTA-3PRGD2 was evaluated in U87MG tumor-bearing nude mice. The U87MG tumor uptake of ¹¹¹In-DOTA-3PRGD2 was slightly lower than that of the ¹¹¹In-DOTA-RGD4 (e.g., 6.13 ± 0.82%ID/g vs 6.43 ± 1.6%ID/g at 4 h postinjection), but the uptake of ¹¹¹In-DOTA-3PRGD2 in normal organs, such as liver and kidneys, was much lower than that of ¹¹¹In-DOTA-RGD4, which resulted in much higher tumor-to-nontumor ratios and lower toxicity. The MTD of ⁹⁰Y-DOTA-RGD4 in nude mice is less than 44.4 MBq, while the MTD of ⁹⁰Y-DOTA-3PRGD2 in mice is more than 55.5 MBq. ⁹⁰Y-DOTA-3PRGD2 administration exhibited a similar tumor inhibition effect as compared with ⁹⁰Y-DOTA-RGD4 at the same dose. The tumor vasculature in the ⁹⁰Y-DOTA-3PRGD2 treatment group was much less than the control groups. Radionuclide therapy studies exhibited that both ⁹⁰Y-DOTA-RGD4 and ⁹⁰Y-DOTA-3PRGD2 caused significant tumor growth delay in the U87MG tumor model. Compared to ⁹⁰Y-DOTA-RGD4, the low accumulation of ⁹⁰Y-DOTA-3PRGD2 in normal organs led to lower toxicity and higher MTD in nude mice, which would make it more suitable for high dose or multiple-dose regimens, in order to achieve maximum therapeutic efficacy.

64Cu Labeled AmBaSar-RGD2 for micro-PET Imaging of Integrin αvβ3 Expression

Integrin αvβ3 plays a critical role in tumor-induced angiogenesis and metastasis. Previously, a 64Cu-AmBaSar- RGD monomer with high in vivo stability compared with 64Cu-DOTA-RGD was developed for integrin αvβ3 PET imaging. It has been established that dimeric RGD peptides have higher receptor-binding affinity and superior in vivo kinetics compared with monomeric RGD peptides due to the polyvalency effect. In this context, we synthesized and evaluated 64Cu-labeled AmBaSar dimeric RGD conjugates (64Cu-AmBaSar-RGD2) for PET imaging of integrin αvβ3 expression. The dimeric RGD peptide was conjugated with a cage-like chelator AmBaSar and labeled with 64Cu. Cell binding, microPET imaging, receptor blocking, and biodistribution studies of 64Cu-AmBaSar-RGD2 were conducted in the U87MG human glioblastoma xenograft model. AmBaSar-RGD2 conjugate was obtained in reasonable yield (45.0 ± 2.5%, n= 4) and the identity was confirmed by HPLC and MS (found 1779.8, calculated m/z for [M+H]+ M: C81H125N27O19 1779.9). 64Cu-AmBaSar-RGD2 was obtained with high radiochemical yield (92.0 ± 1.3%) and purity (≥ 98.0%) under mild conditions (pH 5.0~5.5, 23~37 °C) in 30 min. The specific activity of 64Cu-AmBaSar-RGD2 was estimated to be 15-22 GBq/µmol at the end of synthesis. Based on microPET imaging and biodistribution studies, 64Cu-AmBaSar-RGD2 has demonstrated higher tumor uptake at selected time points than 64Cu-AmBaSar-RGD. At 20 h p.i., the tumor uptake reached 0.65 ± 0.05 %ID/g for 64Cu-AmBaSar-RGD and 1.76 ± 0.38 %ID/g for 64Cu-AmBaSar-RGD2, respectively. The integrin αvβ3 targeting specificity was confirmed by blocking experiments. Therefore, the new tracer 64Cu-AmBaSar- RGD2 exhibited better tumor-targeting efficacy and more favorable in vivo pharmacokinetics than the 64Cu labeled RGD monomer due to the polyvalency effect.

New Methods for Labeling RGD Peptides with Bromine-76.

Direct bromination of the tyrosine residues of peptides and antibodies with bromine-76, to create probes for PET imaging, has been reported. For peptides that do not contain tyrosine residues, however, a prosthetic group is required to achieve labeling via conjugation to other functional groups such as terminal α-amines or lysine ε-amines. The goal of this study was to develop new strategies for labeling small peptides with Br-76 using either a direct labeling method or a prosthetic group, depending on the available functional group on the peptides. A new labeling agent, N-succinimidyl-3-[(76)Br]bromo-2,6-dimethoxybenzoate ([(76)Br]SBDMB) was prepared for cyclic RGD peptide labeling. N-succinimidyl-2, 6-dimethoxybenzoate was also used to pre-attach a 2, 6-dimethoxybenzoyl (DMB) moiety to the peptide, which could then be labeled with Br-76. A competitive cell binding assay was performed to determine the binding affinity of the brominated peptides. PET imaging of U87MG human glioblastoma xenografted mice was performed using [(76)Br]-BrE[c(RGDyK)](2) and [(76)Br]-BrDMB-E[c(RGDyK)](2). An ex vivo biodistribution assay was performed to confirm PET quantification. The mechanisms of bromination reaction between DMB-c(RGDyK) and the brominating agent CH(3)COOBr were investigated with the SCRF-B3LYP/6-31G* method with the Gaussian 09 program package. The yield for direct labeling of c(RGDyK) and E[c(RGDyK)](2) using chloramine-T and peracetic acid at ambient temperature was greater than 50%. The yield for [(76)Br]SBDMB was over 60% using peracetic acid. The conjugation yields for labeling c(RGDfK) and c(RGDyK) were over 70% using the prosthetic group at room temperature. Labeling yield for pre-conjugated peptides was over 60%. SDMB conjugation and bromination did not affect the binding affinity of the peptides with integrin receptors. Both [(76)Br]Br-E[c(RGDyK)](2) and [(76)Br]BrDMB-E[c(RGDyK)](2) showed high tumor uptake in U87MG tumor bearing mice. The specificity of the imaging tracers was confirmed by decreased tumor uptake after co-administration of unlabeled dimeric RGD peptides. The energy barrier of the transition state of bromination for the dimethoxybenzoyl group was about 9 kcal/mol lower than that for the tyrosine residue. In conclusion, the newly developed N-succinimidyl-2, 6-dimethoxybenzoate molecule can be used either for one step labeling through pre-conjugation or as the precursor for a Br-76 labeled prosthetic group for indirect labeling. Radiobromination on a dimethoxybenzoyl group has selectivity over radiobromination on tyrosine. The energy barrier difference of the transition states of bromination between the dimethoxybenzoyl group and the tyrosine residue may account for the reaction selectivity when both groups are present in the same molecule.

PET imaging of αvβ3 integrin expression in tumours with 68Ga-labelled mono-, di- and tetrameric RGD peptides

PurposeDue to the restricted expression of αvβ3 in tumours, αvβ3 is considered a suitable receptor for tumour targeting. In this study the αvβ3-binding characteristics of 68Ga-labelled monomeric, dimeric and tetrameric RGD peptides were determined and compared with their 111In-labelled counterparts.MethodsA monomeric (E-c(RGDfK)), a dimeric (E-[c(RGDfK)]2) and a tetrameric (E{E[c(RGDfK)]2}2) RGD peptide were synthesised, conjugated with DOTA and radiolabelled with 68Ga. In vitro αvβ3-binding characteristics were determined in a competitive binding assay. In vivo αvβ3-targeting characteristics of the compounds were assessed in mice with subcutaneously growing SK-RC-52 xenografts. In addition, microPET images were acquired using a microPET/CT scanner.ResultsThe IC50 values for the Ga(III)-labelled DOTA-E-c(RGDfK), DOTA-E-[c(RGDfK)]2 and DOTA-E{E[c(RGDfK)]2}2 were 23.9 ± 1.22, 8.99 ± 1.20 and 1.74 ± 1.18 nM, respectively, and were similar to those of the In(III)-labelled mono-, di- and tetrameric RGD peptides (26.6 ± 1.15, 3.34 ± 1.16 and 1.80 ± 1.37 nM, respectively). At 2 h post-injection, tumour uptake of the 68Ga-labelled mono-, di- and tetrameric RGD peptides (3.30 ± 0.30, 5.24 ± 0.27 and 7.11 ± 0.67%ID/g, respectively) was comparable to that of their 111In-labelled counterparts (2.70 ± 0.29, 5.61 ± 0.85 and 7.32 ± 2.45%ID/g, respectively). PET scans were in line with the biodistribution data. On all PET scans, the tumour could be clearly visualised.ConclusionThe integrin affinity and the tumour uptake followed the order of DOTA-tetramer > DOTA-dimer > DOTA-monomer. The 68Ga-labelled tetrameric RGD peptide has excellent characteristics for imaging of αvβ3 expression with PET.Electronic supplementary materialThe online version of this article (doi:10.1007/s00259-010-1615-x) contains supplementary material, which is available to authorized users.

Blood Clearance Kinetics, Biodistribution, and Radiation Dosimetry of a Kit-Formulated Integrin αvβ3-Selective Radiotracer 99mTc-3PRGD2 in Non-Human Primates

(99m)Tc-3PRGD(2) is a (99m)Tc-labeled dimeric cyclic RGD peptide with increased receptor binding affinity and improved kinetics for in vivo imaging of integrin α(v)β(3) expression in nude mouse model. To accelerate its clinical translation, we reported here the evaluation of the kit-formulated (99m)Tc-3PRGD(2) in healthy cynomolgus primates for its blood clearance kinetics, biodistribution, and radiation dosimetry. Healthy cynomolgus primates (4.1 ± 0.7 kg, n = 5) were anesthetized, and the venous blood samples were collected via a femoral vein catheter at various time points after injection of ~555 MBq of (99m)Tc-3PRGD(2). Serial whole-body scans were performed with a dual-head single photon emission computed tomography system after administering ~555 MBq of (99m)Tc-3PRGD(2) in the non-human primates, and the radiation dosimetry estimate was calculated. (99m)Tc-3PRGD(2) could be easily obtained from freeze-dried kits with high radiochemical purity (>95%) and high specific activity (~5 Ci/μmol). (99m)Tc-3PRGD(2) had a rapid blood clearance with less than 1% of the initial radioactivity remaining in the blood circulation at 60 min postinjection. No adverse reactions were observed up to 4 weeks after the repeated dosing. The whole-body images exhibited high kidney uptake of (99m)Tc-3PRGD(2) and high radioactivity accumulation in the bladder, demonstrating the rapid renal clearance of this tracer. The highest radiation doses of (99m)Tc-3PRGD(2) were found in the kidneys (13.2 ± 1.08 μGy/MBq) and the bladder wall (33.1 ± 1.91 μGy/MBq). (99m)Tc-3PRGD(2) can be readily available using the kit formulation. This tracer is safe and well tolerated, and no adverse events occurred in non-human primates. Further clinical testing and translation of (99m)Tc-3PRGD(2) for noninvasive imaging of integrin α(v)β(3) in humans are warranted.

Multivalent cyclic RGD ligands: influence of linker lengths on receptor binding

Peptides involving the RGD motive (arginine-glycine-aspartic acid) recognize members of the integrin receptor family. Since the receptors are located mainly on the surface of endothelial cells, structural modifications including multimers of c(RGDfE) were recently found to improve the binding avidity for α(v)β(3) integrin significantly. The multivalent RGD peptides exhibited rather loose linkages partly including oligo(ethylene glycol) spacers (EG(n)) with different chain lengths. Therefore, the dependence of multivalent RGD systems with and without EG(n) linkers were investigated on their binding properties to cultured α(v)β(3) integrin-expressing U87MG cells. We synthesized a series of di-, tri- and tetravalent rigid scaffolds (terephthalic acid, trimesic acid and adamantane-1,3,5,7-tetracarboxylic acid) conjugated to c(RGDyK) ligands, which were linked contiguously or separated by the oligo(ethylene glycol) spacers. The inhibition constants of these c(RGDyK) derivatives were determined by competition assays with (125)I-labeled echistatin. While c(RGDyK) function is a relative weak competitor against [(125)I]echistatin (K(i), 329 ± 18 nM) for α(v)β(3) integrin-expressing U87MG cells, RGD dimers improved the competition potency considerably (K(i), 64 ± 23 nM). This effect was even more pronounced with the RGD trimers (K(i), 40 ± 7 nM) and tetramers (K(i), 26±9 nM). The introduction of EG(n) spacers and the increase of linker lengths proved to be detrimental since more competitors were needed to compete with [(125)I]echistatin. The EG(6) group, for example, reduced the inhibition constants by 29% (dimer), 57% (trimer) and 97% (tetramer). The binding experiments performed with the three forms of multivalent RGD ligands indicate the weakening of competitive potency against [(125)I]echistatin with the introduction of EG(n) spacers. This effect may be related to the decrease of the effective RGD molarity, which becomes most prominent within the tetravalent series.

Synergistic effects of tethered growth factors and adhesion ligands on DNA synthesis and function of primary hepatocytes cultured on soft synthetic hydrogels

The composition, presentation, and spatial orientation of extracellular matrix molecules and growth factors are key regulators of cell behavior. Here, we used self-assembling peptide nanofiber gels as a modular scaffold to investigate how fibronectin-derived adhesion ligands and different modes of epidermal growth factor (EGF) presentation synergistically regulate multiple facets of primary rat hepatocyte behavior in the context of a soft gel. In the presence of soluble EGF, inclusion of dimeric RGD and the heparin binding domain from fibronectin (HB) increased hepatocyte aggregation, spreading, and metabolic function compared to unmodified gels or gels modified with a single motif, but unlike rigid substrates, gels failed to induce DNA synthesis. Tethered EGF dramatically stimulated cell aggregation and spreading under all adhesive ligand conditions and also preserved metabolic function. Surprisingly, tethered EGF elicited DNA synthesis on gels with RGD and HB. Phenotypic differences between soluble and tethered EGF stimulation of cells on peptide gels are correlated with differences in expression and phosphorylation the EGF receptor and its heterodimerization partner ErbB2, and activation of the downstream signaling node ERK1/2. These modular matrices reveal new facets of hepatocellular biology in culture and may be more broadly useful in culture of other soft tissues.

Optical Imaging of Integrin αvβ3Expression with Near-Infrared Fluorescent RGD Dimer with Tetra(ethylene glycol) Linkers

Integrin alphavbeta3 plays great roles in tumor angiogenesis, invasion, and metastasis. We report here the noninvasive visualization of tumor integrin alphavbeta3 expression by using near-infrared fluorescence (NIRF) imaging of an IRDye800-labeled new cyclic RGD (arginine-glycine-aspartic acid) dimer with tetra(ethylene glycol) (PEG4) linkers (ie, E[PEG4-c(RGDfK)]2, PEG4 = 15-amino-4,7,10,13-tetraoxapentadecanoic acid) in a U87MG tumor model. Fluorescent dye-labeled E[PEG4-c(RGDfK)]2 were subjected to in vitro cell staining, in vivo NIRF imaging, ex vivo NIRF imaging, and histologic studies. The in vitro and in vivo characterization of dye-labeled E[PEG4-c(RGDfK)]2 were compared with dye-labeled RGD dimer without PEG4 linkers (namely, E[c(RGDfK)]2). Both Cy5.5-E[PEG4-c(RGDfK)]2 and Cy5.5-E[c(RGDfK)]2 exhibited integrin alphavbeta3 binding specificity in a cell-staining experiment. In vivo NIRF imaging showed higher tumor accumulation and tumor to background contrast of IRDye800-E[PEG4-c(RGDfK)]2 over IRDye800-E[c(RGDfK)]2. The tumor integrin alphavbeta3 specificity of IRDye800-E[PEG4-c(RGDfK)]2 was confirmed by successful inhibition of tumor uptake in the presence of an excess dose of c(RGDfK). Histologic examination revealed both tumor vasculature and tumor cell integrin alphavbeta3 binding of IRDye800-E[PEG4-c(RGDfK)]2 in vivo. In summary, NIRF imaging with IRDye800-E[PEG4-c(RGDfK)]2 offers an easy, fast, and low-cost way to detect and semiquantify tumor integrin alphavbeta3 expression in living subjects.

18F-Labeled Galacto and PEGylated RGD Dimers for PET Imaging of αvβ3 Integrin Expression

In vivo imaging of α(v)β(3) has important diagnostic and therapeutic applications. (18)F-Galacto-arginine-glycine-aspartic acid (RGD) has been developed for positron emission tomography (PET) imaging of integrin α(v)β(3) expression and is now being tested on humans. Dimerization and multimerization of cyclic RGD peptides have been reported to improve the integrin α(v)β(3)-binding affinity due to the polyvalency effect. Here, we compared a number of new dimeric RGD peptide tracers with the clinically used (18)F-galacto-RGD. RGD monomers and dimers were coupled with galacto or PEG(3) linkers, and labeled with (18)F using 4-nitrophenyl 2-(18)F-fluoropropionate ((18)F-NFP) or N-succinimidyl 4-(18)F-fluorobenzoate as a prosthetic group. The newly developed tracers were evaluated by cell-based receptor-binding assay, biodistribution, and small-animal PET studies in a subcutaneous U87MG glioblastoma xenograft model. Starting with (18)F-F(-), the total reaction time for (18)F-FP-SRGD2 and (18)F-FP-PRGD2 is about 120 min. The decay-corrected radiochemical yields for (18)F-FP-SRGD2 and (18)F-FP-PRGD2 are 52 ± 9% and 80 ± 7% calculated from (18)F-NFP. Noninvasive small-animal PET and direct tissue sampling experiments demonstrated that the dimeric RGD peptides had significantly higher tumor uptake as compared to (18)F-galacto-RGD. Dimeric RGD peptide tracers with relatively high tumor integrin-specific accumulation and favorable in vivo kinetics may have the potential to be translated into clinic for integrin α(v)β(3) imaging.

2-Mercaptoacetylglycylglycyl (MAG2) as a bifunctional chelator for 99mTc-labeling of cyclic RGD dimers: effect of technetium chelate on tumor uptake and pharmacokinetics.

This report describes the synthesis of MAG(2)-PEG(4)-E[c(RGDfK)](2) (MAG(2)-P-RGD(2): MAG(2) = S-benzoylmercaptoacetylglycylglycyl; PEG(4) = 15-amino-4,7,10,13-tetraoxapentadecanoic acid) and MAG(2)-PEG(4)-E[PEG(4)-c(RGDfK)](2) (MAG(2)-3P-RGD(2)), and the evaluation of (99m)TcO(MAG(2)-P-RGD(2)) and (99m)TcO(MAG(2)-3P-RGD(2)) as new radiotracers for tumor imaging in the athymic nude mice bearing U87MG human glioma xenografts. We found that MAG(2) is such an efficient bifunctional chelating agent that (99m)TcO(MAG(2)-P-RGD(2)) and (99m)TcO(MAG(2)-3P-RGD(2)) coul d be prepared in high yield (>90%) with high specific activity (∼5 Ci/μmol) using a kit formulation. (99m)TcO(MAG(2)-P-RGD(2)) and (99m)TcO(MAG(2)-3P-RGD(2)) have very high solution stability in the kit matrix. Biodistribution data in athymic nude mice bearing U87MG human glioma xenografts indicated that replacing the highly charged [(99m)Tc(HYNIC = 6-hydrazinonicotinyl and TPPTS = trisodium triphenylphosphine-3,3',3''-trisulfonate) with smaller (99m)TcO(MAG(2)) resulted in a significant increase in the radiotracer uptake in the tumor and normal organs most likely due to the higher lipophilicity of (99m)TcO(MAG(2)-3P-RGD(2)) (log P = -3.15 ± 0.10) than that for [(99m)Tc(HYNIC-3P-RGD(2))(tricine)(TPPTS)] ((99m)Tc-3P-RGD(2): log P = -3.96 ± 0.05). Even though (99m)TcO(MAG(2)-3P-RGD(2)) has better tumor uptake (15.36 ± 2.17 %ID/g at 60 min postinjection (p.i.)) than (99m)Tc-3P-RGD(2) (9.15 ± 2.13 %ID/g at 60 min p.i.), its tumor-to-background (T/B) ratios (tumor/blood = 13.52 ± 4.57; tumor/liver = 4.25 ± 0.88; tumor/lung = 3.17 ± 0.60; and tumor/muscle = 8.34 ± 2.34) are not as good as those of (99m)Tc-3P-RGD(2) (tumor/blood = 36.0 ± 11.5; tumor/liver = 5.14 ± 1.46; tumor/lung = 4.36 ± 0.54; and tumor/muscle = 13.70 ± 2.21) at 60 min p.i. On the basis of these results, we believe that (99m)Tc-3P-RGD(2) remains a better radiotracer because of its higher T/B ratios.

99mTcO(MAG2-3G3-dimer): a new integrin αvβ3-targeted SPECT radiotracer with high tumor uptake and favorable pharmacokinetics

This report presents the synthesis of a cyclic RGD dimer conjugate, MAG(2)-G(3)-E[G(3)-c(RGDfK)](2) (MAG(2)-3G(3)-dimer, G(3) = Gly-Gly-Gly, MAG(2) = S-benzoyl mercaptoacetylglycylglycyl), and evaluation of its (99m)Tc complex, (99m)TcO(MAG(2)-3G(3)-dimer), as a new radiotracer for imaging the tumor integrin alpha(v)beta(3) expression. An in vitro displacement assay was used to determine the integrin alpha(v)beta(3) binding affinity of MAG(2)-3G(3)-dimer against (125)I-c(RGDyK) bound to U87MG human glioma cells. The athymic nude mice bearing U87MG glioma xenografts were used for biodistribution and planar imaging studies. We found that (1) MAG(2) is such a highly effective bifunctional chelator that (99m)TcO(MAG(2)-3G(3)-dimer) can be prepared in high yield (radiochemical purity >95%) and with high specific activity ( approximately 5 Ci/micromol) using a kit formulation; (2) (99m)TcO(MAG(2)-3G(3)-dimer) has very high solution stability in the kit matrix; and (3) (99m)TcO(MAG(2)-3G(3)-dimer) has very fast clearance kinetics from the intestine, liver, and kidneys. Among the (99m)Tc-labeled cyclic RGD peptides evaluated in the xenografted U87MG glioma models, (99m)TcO(MAG(2)-3G(3)-dimer) has the best pharmacokinetics and tumor to background ratios (tumor/liver = 4.29 +/- 1.00 at 30 min postinjection and 8.29 +/- 1.50 at 120 min postinjection; tumor/kidney = 1.16 +/- 0.19 at 30 min postinjection and 2.49 +/- 0.25 at 120 min postinjection). Planar imaging studies showed that tumors in the glioma-bearing mice administered with (99m)TcO(MAG(2)-3G(3)-dimer) can be visualized with excellent contrast as early as 15 min postinjection. (99m)TcO(MAG(2)-3G(3)-dimer) was able to maintain its chemical integrity in kidneys (>80% intact) and liver (>95% intact) over the 2-h period. However, there was significant metabolism (>50% of the injected radioactivity) detected in both urine and feces samples. (99m)TcO(MAG(2)-3G(3)-dimer) is a very attractive radiotracer for early detection of integrin alpha(v)beta(3)-positive tumors and has significant advantages over the (18)F-labeled RGD peptide radiotracers with respect to the cost, availability, and easiness for routine clinical preparation.

Noninvasive imaging of tumor integrin expression using 18F-labeled RGD dimer peptide with PEG4 linkers

Various radiolabeled Arg-Gly-Asp (RGD) peptides have been previously investigated for tumor integrin alpha(v)beta(3) imaging. To further develop RGD radiotracers with enhanced tumor-targeting efficacy and improved in vivo pharmacokinetics, we designed a new RGD homodimeric peptide with two PEG(4) spacers (PEG(4) = 15-amino-4,7,10,13-tetraoxapentadecanoic acid) between the two monomeric RGD motifs and one PEG(4) linker on the glutamate alpha-amino group ((18)F-labeled PEG(4)-E[PEG(4)-c(RGDfK)](2), P-PRGD2), as a promising agent for noninvasive imaging of integrin expression in mouse models. P-PRGD2 was labeled with (18)F via 4-nitrophenyl 2-(18)F-fluoropropionate ((18)F-FP) prosthetic group. In vitro and in vivo characteristics of the new dimeric RGD peptide tracer (18)F-FP-P-PRGD2 were investigated and compared with those of (18)F-FP-P-RGD2 ((18)F-labeled RGD dimer without two PEG(4) spacers between the two RGD motifs). The ability of (18)F-FP-P-PRGD2 to image tumor vascular integrin expression was evaluated in a 4T1 murine breast tumor model. With the insertion of two PEG(4) spacers between the two RGD motifs, (18)F-FP-P-PRGD2 showed enhanced integrin alpha(v)beta(3)-binding affinity, increased tumor uptake and tumor-to-nontumor background ratios compared with (18)F-FP-P-RGD2 in U87MG tumors. MicroPET imaging with (18)F-FP-P-PRGD2 revealed high tumor contrast and low background in tumor-bearing nude mice. Biodistribution studies confirmed the in vivo integrin alpha(v)beta(3)-binding specificity of (18)F-FP-P-RGD2. (18)F-FP-P-PRGD2 can specifically image integrin alpha(v)beta(3) on the activated endothelial cells of tumor neovasculature. (18)F-FP-P-PRGD2 can provide important information on integrin expression on the tumor vasculature. The high integrin binding affinity and specificity, excellent pharmacokinetic properties and metabolic stability make the new RGD dimeric tracer (18)F-FP-P-PRGD2 a promising agent for PET imaging of tumor angiogenesis and for monitoring the efficacy of antiangiogenic treatment.

68Ga-labeled cyclic RGD dimers with Gly3 and PEG4 linkers: promising agents for tumor integrin αvβ3 PET imaging

Radiolabeled cyclic RGD (Arg-Gly-Asp) peptides have great potential for the early tumor detection and noninvasive monitoring of tumor metastasis and therapeutic response. (18)F-labeled RGD analogs ([(18)F]-AH111585 and [(18)F]Galacto-RGD) have been investigated in clinical trials for positron emission tomography (PET) imaging of integrin expression in cancer patients. To develop new RGD radiotracers with higher tumor accumulation, improved in vivo kinetics, easy availability and low cost, we developed two new RGD peptides and labeled them with generator-eluted (68)Ga (t(1/2) = 68 min) for PET imaging of integrin alpha(v)beta(3) expression in tumor xenograft models. The two new cyclic RGD dimers, E[PEG(4)-c(RGDfK)](2) (P(4)-RGD2, PEG(4) = 15-amino-4,7,10,13-tetraoxapentadecanoic acid) and E[Gly(3)-c(RGDfK)](2) (G(3)-RGD2, G(3) = Gly-Gly-Gly) were designed, synthesized and conjugated with 1,4,7-triazacyclononanetriacetic acid (NOTA) for (68)Ga labeling. The microPET imaging and biodistribution of the (68)Ga labeled RGD tracers were investigated in integrin alpha(v)beta(3)-positive tumor xenografts. The new RGD dimers with the Gly(3) and PEG(4) linkers showed higher integrin alpha(v)beta(3) binding affinity than no-linker RGD dimer (RGD2). NOTA-G(3)-RGD2 and NOTA-P(4)-RGD2 could be labeled with (68)Ga within 30 min with higher purity (>98%) and specific activity (8.88-11.84 MBq/nmol). Both (68)Ga-NOTA-P(4)-RGD2 and (68)Ga-NOTA-G(3)-RGD2 exhibited significantly higher tumor uptake and tumor-to-normal tissue ratios than (68)Ga-NOTA-RGD2. Because of their high affinity, high specificity and excellent pharmacokinetic properties, further investigation of the two novel RGD dimers for clinical PET imaging of integrin alpha(v)beta(3) expression in cancer patients is warranted.

Improving tumor-targeting capability and pharmacokinetics of (99m)Tc-labeled cyclic RGD dimers with PEG(4) linkers.

This report describes the synthesis of two cyclic RGD (Arg-Gly-Asp) conjugates, HYNIC-2PEG(4)-dimer (HYNIC = 6-hydrazinonicotinyl; 2PEG(4)-dimer = E[PEG(4)-c(RGDfK)](2); and PEG(4) = 15-amino-4,7,10,13-tetraoxapentadecanoic acid) and HYNIC-3PEG(4)-dimer (3PEG(4)-dimer = PEG(4)-E[PEG(4)-c(RGDfK)](2)), and evaluation of their (99m)Tc complexes [(99m)Tc(HYNIC-2PEG(4)-dimer)(tricine)(TPPTS)] ((99m)Tc-2PEG(4)-dimer: TPPTS = trisodium triphenylphosphine-3,3',3''-trisulfonate) and [(99m)Tc(HYNIC-3PEG(4)-dimer)(tricine)(TPPTS)] ((99m)Tc-3PEG(4)-dimer) as novel radiotracers for imaging integrin alpha(v)beta(3) expression in athymic nude mice bearing U87MG glioma and MDA-MB-435 breast cancer xenografts. The integrin alpha(v)beta(3) binding affinities of RGD peptides were determined by competitive displacement of (125)I-c(RGDyK) on U87MG glioma cells. It was found that the two PEG(4) linkers between RGD motifs in HYNIC-2PEG(4)-dimer (IC(50) = 2.8 +/- 0.5 nM) and HYNIC-3PEG(4)-dimer (IC(50) = 2.4 +/- 0.7 nM) are responsible for their higher integrin alpha(v)beta(3) binding affinity than that of HYNIC-PEG(4)-dimer (PEG(4)-dimer = PEG(4)-E[c(RGDfK)](2); IC(50) = 7.5 +/- 2.3 nM). Addition of extra PEG(4) linker in HYNIC-3PEG(4)-dimer has little impact on integrin alpha(v)beta(3) binding affinity. (99m)Tc-2PEG(4)-dimer and (99m)Tc-3PEG(4)-dimer were prepared in high yield with >95% radiochemical purity and the specific activity of >10 Ci/mumol. Biodistribution studies clearly demonstrated that PEG(4) linkers are particularly useful for improving the tumor uptake and clearance kinetics of (99m)Tc-2PEG(4)-dimer and (99m)Tc-3PEG(4)-dimer from noncancerous organs. It was also found that there was a linear relationship between the tumor size and radiotracer tumor uptake expressed as %ID (percentage of the injected dose) in U87MG glioma and MDA-MB-435 breast tumor models. The blocking experiment showed that the tumor uptake of (99m)Tc-2PEG(4)-dimer is integrin alpha(v)beta(3)-mediated. In the metabolism study, (99m)Tc-2PEG(4)-dimer had high metabolic stability during its excretion from renal and hepatobiliary routes. (99m)Tc-3PEG(4)-dimer also remained intact during thee excretion from the renal route, but, had approximately 30% metabolism during the excretion from the hepatobiliary route. Planar imaging studies in U87MG glioma and MDA-MB-435 breast tumor models showed that the tumors of approximately 5 mm in diameter could be readily visualized with excellent contrast. Thus, (99m)Tc-3PEG(4)-dimer is a very promising radiotracer for the early detection of integrin alpha(v)beta(3)-positive tumors, and may have the potential for noninvasive monitoring of tumor growth or treatment efficacy.