Dimeric RGD Peptide Research Articles

SP-073 - Direct 18F-labeling of a dimeric RGD peptide via F-nucleophilic substitution on a phosphonate prothesis

SP-073 - Direct 18F-labeling of a dimeric RGD peptide via F-nucleophilic substitution on a phosphonate prothesis

An improved kit formulation for one-pot synthesis of [99m Tc]Tc-HYNIC-E[c(RGDfK)]2 for routine clinical use in cancer imaging.

Radiolabeled Arg-Gly-Asp (RGD) peptide derivatives have immense potential for non-invasive monitoring of malignancies overexpressing integrin αv β3 receptors. Easy availability of suitable radiotracers would augment the utility of this class of molecular imaging agents. Towards this, the present article describes the development of an improved lyophilized kit for the routine clinical formulation of [99m Tc]Tc complex of HYNIC-conjugated dimeric cyclic RGD peptide derivative E-[c(RGDfK)]2 (E = glutamic acid, f = phenyl alanine, K = lysine) without using Sn2+ and systematic evaluation of its efficacy. Five batches of the kits were prepared, and [99m Tc]Tc-HYNIC-E[c(RGDfK)]2 radiotracer was synthesized with high radiochemical purity (98.6 ± 0.5%) and specific activity (124.8 GBq/μmol) using the kits. Biodistribution studies in C57BL/6 mice bearing melanoma tumor exhibited significant accumulation of the radiotracer in tumor (5.32 ± 0.56 %ID/g at 60 min p.i.), and this uptake was also found to be receptor-specific by blocking studies. Preliminary human clinical investigations carried out in 10 breast cancer patients revealed high radiotracer uptake in the tumor along with good tumor-to-background contrast. The developed kit formulation showed an exceptionally high shelf-life of at least 18 months. These results demonstrated promising attributes of the developed kit formulation and warrant more extensive clinical investigations.

68Ga-labeled dimeric and trimeric cyclic RGD peptides as potential PET radiotracers for imaging gliomas

68Ga-labeled cyclic RGD dimers and trimer were evaluated as PET radiotracers. It was found that the linker group had little impact on αvβ3 binding affinity of RGD dimers, which share similar αvβ3 binding affinity with the RGD trimer despite of their different multiplicity. Biodistribution properties of 68Ga radiotracers depend on RGD peptides and radiometal chelates. Among the 68Ga radiotracers evaluated, 68Ga-I2P-RGD2 has the best tumor uptake with good tumor-to-background ratios, and is a good PET radiotracer for imaging gliomas.

Birnessite: A New‐Generation and Cost Effective Ion Exchange Material for Separation of Clinical‐Grade 90Y from 90Sr/90Y Mixture

AbstractThe widespread clinical utilization of 90Y for preparation of target specific radiopharmaceuticals stipulates design of superior advanced materials for facile and cost effective separation of 90Y. We demonstrate for the first time potential of birnessite type phylomanganates for radiochemical separation of clinical grade 90Y from 90Sr/90Y mixture. Sodium birnessite was synthesized by controlled oxidation of MnCl2.4H2O in presence of excess of NaOH and characterized thoroughly for structure, microstructure and purity. Ion exchange experiments in the presence of 90Sr/90Y equilibrium mixture confirmed the exceptionally high selectivity of synthetic sodium birnessite for exchanging Sr2+ ions and the conditions have been optimized to achieve radiochemically pure 90Y (> 80% separation yield) meeting all the pharmaceutical requirements for its clinical use. Radionuclidic purity of 90Y could be achieved to < 1 × 10−4 % of 90Sr which is well below the limit set internationally (2 × 10−3 %). The separated 90Y has been used to prepare 90Y radiolabeled 1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetic acid coupled dimeric cyclic RGD peptide derivative [DOTA−E[c(RGDfK)]2] and the biological efficacy of the radiotracer was established in C57/BL6 mice bearing melanoma tumors. The results indicated highly target specific radiotherapy suggesting the potential of sodium birnessite for cost effective radiochemical separation of 90Y.

Automated preparation of 2-[18 F]fluoropropionate labeled peptides using a flexible, multi-stage synthesis platform (iPHASE Flexlab).

Radiolabelled peptides are vital tools used in positron emission tomography imaging for the diagnosis of disease, drug discovery, and biomedical research. Peptides are typically labeled through conjugation to a radiolabelled prosthetic group, which usually necessitates complex, multi-step procedures, especially for fluorine-18 labeled peptides. Herein, we describe the automated synthesis and formulation of 2-[18 F]fluoropropionate labeled RGD-peptides through use of the iPHASE Flexlab as an effective dual-stage radiochemical synthesis module. The fully automated preparation of the monomeric RGD-peptides, [18 F]FP-GalactoRGD and [18 F]FP-c(RGDy(SO3 )K), was accomplished in under 90minutes with n.d.c. radiochemical yields ca. 7% from fluoride. Similarly, the automated preparation of the dimeric RGD-peptides, [18 F]F-PRGD2 and [18 F]FP-E(RGDy(SO3 )K)2 , was accomplished in under 105minutes with n.d.c. yields ca. 4% from fluoride.

Iminodiacetic acid as bifunctional linker for dimerization of cyclic RGD peptides

IntroductionIn this study, I2P-RGD2 was used as the example to illustrate a novel approach for dimerization of cyclic RGD peptides. The main objective of this study was to explore the impact of bifunctional linkers (glutamic acid vs. iminodiacetic acid) on tumor-targeting capability and excretion kinetics of the 99mTc-labeled dimeric cyclic RGD peptides. MethodsHYNIC-I2P-RGD2 was prepared by reacting I2P-RGD2 with HYNIC-OSu in the presence of diisopropylethylamine, and was evaluated for its αvβ3 binding affinity against 125I-echistatin bound to U87MG glioma cells. 99mTc-I2P-RGD2 was prepared with high specific activity (~185GBq/μmol). The athymic nude mice bearing U87MG glioma xenografts were used to evaluate its biodistribution properties and image quality in comparison with those of 99mTc-3P-RGD2. ResultsThe IC50 value for HYNIC-I2P-RGD2 was determined to be 39±6nM, which was very close to that (IC50=33±5nM) of HYNIC-3P-RGD2. Replacing glutamic acid with iminodiacetic acid had little impact on αvβ3 binding affinity of cyclic RGD peptides. 99mTc-I2P-RGD2 and 99mTc-3P-RGD2 shared similar tumor uptake values over the 2h period, and its αvβ3-specificity was demonstrated by a blocking experiment. The uptake of 99mTc-I2P-RGD2 was significantly lower than 99mTc-3P-RGD2 in the liver and kidneys. The U87MG glioma tumors were visualized by SPECT with excellent contrast using both 99mTc-I2P-RGD2 and 99mTc-3P-RGD2. ConclusionIminodiacetic acid is an excellent bifunctional linker for dimerization of cyclic RGD peptides. Bifunctional linkers have significant impact on the excretion kinetics of 99mTc radiotracers. Because of its lower liver uptake and better tumor/liver ratios, 99mTc-I2P-RGD2 may have advantages over 99mTc-3P-RGD2 for diagnosis of tumors in chest region.

Preparation and bioevaluation of [99mTcN]2+-labeled tetrameric complex of E-c(RGDfK)2 as a radiotracer for imaging αvβ3 integrins in tumors

The objective of the present work is to radiolabel a dimeric RGD peptide namely, E-c(RGDfK)2 using [99mTcN]2+ core in order to obtain a 99mTc-labeled tetrameric RGD species, which is expected to have an enhanced tumor affinity. 99mTc-labeleld [E-c(RGDfK)2]2 complex was prepared with high radiochemical purity (>95 %). Pharmacokinetic evaluation of the radiolabeled agent, carried out in C57BL/6 mice bearing melanoma tumor, demonstrated good tumor uptake within 30 min of administration (3.94 ± 0.87 % IA/g) and retention therein till 3 h post-injection (2.65 ± 0.31 % IA/g). Blocking experiment revealed the specificity of the complex towards the αvβ3 receptors in the tumor model.

Hybrid PET/optical imaging of integrin αVβ3 receptor expression using a (64)Cu-labeled streptavidin/biotin-based dimeric RGD peptide.

BackgroundHybrid PET/optical imaging provides quantitative and complementary information for diagnosis of tumors. Herein, we developed a 64Cu-labeled AlexaFluor 680-streptavidin ((AF)SAv)/biotin-based dimeric cyclic RGD peptide (RGD2) for hybrid PET/optical imaging of integrin αVβ3 expression.Methods64Cu-1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid (DOTA)-(AF)SAv/biotin-PEG-RGD2 was prepared by formation of a complex comprising DOTA-(AF)SAv and biotin-PEG-RGD2, followed by radiolabeling with 64Cu. Receptor binding studies of DOTA-(AF)SAv/biotin-PEG-RGD2 were performed using U87MG cells and 125I-RGDyK as the radioligand, and cellular uptake studies of 64Cu-DOTA-(AF)SAv/biotin-PEG-RGD2 were also performed. MicroPET imaging followed by optical imaging of U87MG tumor-bearing mice was acquired after injection of the hybrid probe, and region of interest (ROI) analysis of tumors was performed. Ex vivo PET/optical imaging and biodistribution studies of the major tissues were performed after the in vivo imaging, and immunofluorescence staining of the tumor tissue sections was carried out.Results64Cu-DOTA-(AF)SAv/biotin-PEG-RGD2 was prepared in 52.1 ± 5.4 % radiochemical yield and with specific activity of 1.0 ± 0.1 GBq/mg. Receptor binding studies showed that DOTA-(AF)SAv/biotin-PEG-RGD2 had higher binding affinity for integrin αVβ3 than RGD2, reflecting a possible polyvalency effect. Moreover, the hybrid probe revealed time-dependent uptake by U87MG cells. In a microPET/optical imaging study, the hybrid probe demonstrated high accumulation in tumors; ROI analysis revealed 2.7 ± 0.2 % ID/g at 1 h and 4.7 ± 0.2 % ID/g at 21 h after injection, and subsequently acquired optical images showed tumors with strong fluorescence intensity. Ex vivo PET/optical images of the major tissues confirmed the in vivo imaging data, and biodistribution studies demonstrated high and specific uptake in tumors (4.8 ± 0.1 % ID/g). Immunofluorescence staining showed the formation of new blood vessels in tumor tissues, suggesting that the tumor uptake was due to specific binding of the hybrid probe to integrin αVβ3 expressed on tumor cells.ConclusionsThese results indicate that a 64Cu-DOTA-(AF)SAv/biotin-PEG-RGD2 is able to provide quantitative information on hybrid PET/optical imaging of integrin αVβ3 expression.

Toward realization of ‘mix-and-use’ approach in 68Ga radiopharmacy: preparation, evaluation and preliminary clinical utilization of 68Ga-labeled NODAGA-coupled RGD peptide derivative

IntroductionThe present article demonstrates a ‘mix-and-use’ approach for radiolabeling RGD peptide derivative with 68Ga, which is easily adaptable in hospital radiopharmacy practice. The radiotracer thus formulated was successfully used for positron emission tomography (PET) imaging of breast cancer in human patients. MethodsThe conditions for radiolabeling NODAGA-coupled dimeric cyclic RGD peptide derivative [NODAGA-(RGD)2] with 68Ga were optimized using 68Ga obtained from a 68Ge/68Ga generator developed in-house with CeO2-PAN composite sorbent as well as from a commercial 68Ge/68Ga generator obtained from ITG, Germany. Preclinical studies were carried out in C57BL/6 mice bearing melanoma tumors. The radiotracer was prepared in a hospital radiopharmacy using 68Ga obtained from ITG generator and used for monitoring breast cancer patients by positron emission tomography (PET) imaging. Results68Ga-NODAGA-(RGD)2 could be prepared with high radiolabeling yield (>98%) and specific activity (~50GBq/μmol) within 10min at room temperature by mixing 68Ga with the solution of the peptide conjugate. In vivo biodistribution studies showed significant uptake (5.24±0.39% ID/g) in melanoma tumor at 30min post-injection, with high tumor-to-background contrast. The integrin αvβ3 specificity of the tracer was corroborated by blocking study. Preliminary clinical studies in locally advanced breast cancer (LABC) patients indicated specifically high tumor uptake (SUVmax 10–15) with good contrast. ConclusionsThis is one of the very few reports which presents preliminary clinical data on use of 68Ga-NODAGA-(RGD)2 and the developed ‘mix-and-use’ holds tremendous prospect in clinical PET imaging using 68Ga.

Radiolabeled Cyclic RGD Peptide Bioconjugates as Radiotracers Targeting Multiple Integrins.

Angiogenesis is a requirement for tumor growth and metastasis. The angiogenic process depends on vascular endothelial cell migration and invasion, and is regulated by various cell adhesion receptors. Integrins are such a family of receptors that facilitate the cellular adhesion to and migration on extracellular matrix proteins in the intercellular spaces and basement membranes. Among 24 members of the integrin family, αvβ3 is studied most extensively for its role in tumor angiogenesis and metastasis. The αvβ3 is expressed at relatively low levels on epithelial cells and mature endothelial cells, but it is highly expressed on the activated endothelial cells of tumor neovasculature and some tumor cells. This restricted expression makes αvβ3 an excellent target to develop antiangiogenic drugs and diagnostic molecular imaging probes. Since αvβ3 is a receptor for extracellular matrix proteins with one or more RGD tripeptide sequence, many radiolabeled cyclic RGD peptides have been evaluated as "αvβ3-targeted" radiotracers for tumor imaging over the past decade. This article will use the dimeric and tetrameric cyclic RGD peptides developed in our laboratories as examples to illustrate basic principles for development of αvβ3-targeted radiotracers. It will focus on different approaches to maximize the radiotracer tumor uptake and tumor/background ratios. This article will also discuss some important assays for preclinical evaluations of integrin-targeted radiotracers. In general, multimerization of cyclic RGD peptides increases their integrin binding affinity and the tumor uptake and retention times of their radiotracers. Regardless of their multiplicity, the capability of cyclic RGD peptides to bind other integrins (namely, αvβ5, α5β1, α6β4, α4β1, and αvβ6) is expected to enhance the radiotracer tumor uptake due to the increased integrin population. The results from preclinical and clinical studies clearly show that radiolabeled cyclic RGD peptides (such as (99m)Tc-3P-RGD2, (18)F-Alfatide-I, and (18)F-Alfatide-II) are useful as the molecular imaging probes for early cancer detection and noninvasive monitoring of the tumor response to antiangiogenic therapy.

Comparison of biological properties of 111In-labeled dimeric cyclic RGD peptides

IntroductionIn this study two 111In-labeled dimeric cyclic RGD peptides, 111In(DOTA-Galacto-RGD2) and 111In(DOTA-3P-RGD2), were evaluated as radiotracers for breast tumor imaging. The objective was to evaluate the impact of SAA, PEG2 and 1,2,3-triazole linkers as compare to PEG4 on the tumor uptake and excretion kinetics of 111In radiotracers. MethodsDOTA-Galacto-RGD2 was prepared by conjugation of Galacto-RGD2 with DOTA-OSu in the presence of diisopropylethylamine. Its integrin αvβ3 binding affinity was determined using a whole-cell displacement assay against 125I-echistatin bound to U87MG glioma cells, and was compared with those of c(RGDfK), DOTA-3P-RGD2 and DOTA-3P-RGK2 (a nonsense peptide conjugate with “scrambled” RGK sequences). 111In(DOTA-Galacto-RGD2) and 111In(DOTA-3P-RGD2) were prepared and evaluated for their tumor-targeting capability and biodistribution properties in athymic nude mice bearing MDA-MB-435 breast tumor xenografts. Planar imaging studies were performed to demonstrate the utility of 111In(DOTA-Galacto-RGD2) and 111In(DOTA-3P-RGD2) for breast tumor imaging. ResultsIC50 values of DOTA-Galacto-RGD2, DOTA-3P-RGD2, and DOTA-3P-RGK2 were calculated to be 27±2, 29±4, 596±48nM, respectively. The tumor uptake values of 111In(DOTA-Galacto-RGD2) (6.79±0.98, 6.56±0.56, 4.17±0.61 and 1.09±0.13 %ID/g at 1, 4, 24 and 72hours p.i., respectively) were almost identical to those of 111In(DOTA-3P-RGD2) (6.17±1.65, 5.94±0.84, 3.40±0.50 and 0.99±0.20 %ID/g, respectively). 111In(DOTA-Galacto-RGD2) had a faster clearance from blood and muscle than 111In(DOTA-3P-RGD2), leading to higher tumor/blood and tumor/muscle ratios. 111In(DOTA-3P-RGD2) had lower liver uptake and better tumor/liver ratios than 111In(DOTA-Galacto-RGD2). The tumor uptake of 111In(DOTA-Galacto-RGD2) and 111In(DOTA-3P-RGD2) was both integrin αvβ3 and RGD-specific. Imaging data suggest that 111In(DOTA-Galacto-RGD2) and 111In(DOTA-3P-RGD2) are useful as radiotracers for imaging integrin αvβ3-positive breast tumors. ConclusionThe results from this study suggest that replacing PEG4 linkers between two RGD moieties with a pair of SAA, PEG2 and 1,2,3-triazole groups has little impact on integrin αvβ3 binding affinity and tumor uptake of 111In-labeled dimeric cyclic RGD peptides. Despite the subtle differences in their excretion kinetics from noncancerous tissues, 111In(DOTA-Galacto-RGD2) and 111In(DOTA-3P-RGD2) are useful radiotracers for imaging integrin αvβ3-positive breast tumors.

Impact of multiple negative charges on blood clearance and biodistribution characteristics of 99mTc-labeled dimeric cyclic RGD peptides.

This study sought to evaluate the impact of multiple negative charges on blood clearance kinetics and biodistribution properties of 99mTc-labeled RGD peptide dimers. Bioconjugates HYNIC-P6G-RGD2 and HYNIC-P6D-RGD2 were prepared by reacting P6G-RGD2 and P6D-RGD2, respectively, with excess HYNIC-OSu in the presence of diisopropylethylamine. Their IC50 values were determined to be 31 ± 5 and 41 ± 6 nM, respectively, against 125I-echistatin bound to U87MG glioma cells in a whole-cell displacement assay. Complexes [99mTc(HYNIC-P6G-RGD2)(tricine)(TPPTS)] (99mTc-P6G-RGD2) and [99mTc(HYNIC-P6D-RGD2)(tricine)(TPPTS)] (99mTc-P6D-RGD2) were prepared in high radiochemical purity (RCP > 95%) and specific activity (37–110 GBq/μmol). They were evaluated in athymic nude mice bearing U87MG glioma xenografts for their biodistribution. The most significant difference between 99mTc-P6D-RGD2 and 99mTc-P6G-RGD2 was their blood radioactivity levels and tumor uptake. The initial blood radioactivity level for 99mTc-P6D-RGD2 (4.71 ± 1.00%ID/g) was ∼5× higher than that of 99mTc-P6G-RGD2 (0.88 ± 0.05%ID/g), but this difference disappeared at 60 min p.i. 99mTc-P6D-RGD2 had much lower tumor uptake (2.20–3.11%ID/g) than 99mTc-P6G-RGD2 (7.82–9.27%ID/g) over a 2 h period. Since HYNIC-P6D-RGD2 and HYNIC-P6G-RGD2 shared a similar integrin αvβ3 binding affinity (41 ± 6 nM versus 31 ± 5 nM), the difference in their blood activity and tumor uptake is most likely related to the nine negative charges and high protein binding of 99mTc-P6D-RGD2. Despite its low uptake in U87MG tumors, the tumor uptake of 99mTc-P6D-RGD2 was integrin αvβ3-specific. SPECT/CT studies were performed using 99mTc-P6G-RGD2 in athymic nude mice bearing U87MG glioma and MDA-MB-231 breast cancer xenografts. The SPECT/CT data demonstrated the tumor-targeting capability of 99mTc-P6G-RGD2, and its tumor uptake depends on the integrin αvβ3 expression levels on tumor cells and neovasculature. It was concluded that the multiple negative charges have a significant impact on the blood clearance kinetics and tumor uptake of 99mTc-labeled dimeric cyclic RGD peptides.

(18)F-FPPRGD2 PET/CT: pilot phase evaluation of breast cancer patients.

To present data from the first prospective pilot phase trial of breast cancer participants imaged with fluorine 18 ((18)F)-2-fluoropropionyl-labeled PEGylated dimeric arginine-glycine-aspartic acid ( RGD arginine-glycine-aspartic acid ) peptide (PEG3-E[c{ RGD arginine-glycine-aspartic acid yk}]2) ( FPPRGD2 2-fluoropropionyl labeled PEGylated dimeric RGD peptide (PEG3-E[c{RGDyk}]2) ), a radiopharmaceutical agent used in positron emission tomographic (PET) imaging. The local institutional review board approved the HIPAA-compliant protocol. Written informed consent was obtained from each patient. Eight women (age range, 44-67 years; mean age, 54.3 years ± 8.8 [standard deviation]) with newly diagnosed or recurrent breast cancer were recruited between November 2010 and February 2011. (18)F- FPPRGD2 2-fluoropropionyl labeled PEGylated dimeric RGD peptide (PEG3-E[c{RGDyk}]2) PET/computed tomographic (CT) and (18)F-fluorodeoxyglucose ( FDG fluorine 18 fluorodeoxyglucose ) PET/CT examinations were performed within 3 weeks of each other. Dynamic (18)F- FPPRGD2 2-fluoropropionyl labeled PEGylated dimeric RGD peptide (PEG3-E[c{RGDyk}]2) PET and two whole-body static (18)F- FPPRGD2 2-fluoropropionyl labeled PEGylated dimeric RGD peptide (PEG3-E[c{RGDyk}]2) PET/CT scans were obtained. During this time, vital signs and electrocardiograms were recorded at regular intervals. Blood samples were obtained before the injection of (18)F- FPPRGD2 2-fluoropropionyl labeled PEGylated dimeric RGD peptide (PEG3-E[c{RGDyk}]2) and at 24 hours and 1 week after injection to evaluate for toxicity. A nonparametric version of multivariate analysis of variance was used to assess the safety outcome measures simultaneously across time points. A paired two-sample t test was performed to compare the maximum standardized uptake values ( SUVmax maximum standardized uptake value ). (18)F- FPPRGD2 2-fluoropropionyl labeled PEGylated dimeric RGD peptide (PEG3-E[c{RGDyk}]2) was well tolerated, without noticeable changes in vital signs, on electrocardiograms, or in laboratory values. A total of 30 lesions were evaluated at (18)F- FDG fluorine 18 fluorodeoxyglucose PET/CT and (18)F- FPPRGD2 2-fluoropropionyl labeled PEGylated dimeric RGD peptide (PEG3-E[c{RGDyk}]2) PET/CT. The primary breast lesions had (18)F- FPPRGD2 2-fluoropropionyl labeled PEGylated dimeric RGD peptide (PEG3-E[c{RGDyk}]2) uptake with SUVmax maximum standardized uptake value of 2.4-9.4 (mean, 5.6 ± 2.8) 60 minutes after injection, compared with (18)F- FDG fluorine 18 fluorodeoxyglucose uptake with SUVmax maximum standardized uptake value of 2.8-18.6 (mean, 10.4 ± 7.2). Metastatic lesions also showed (18)F- FPPRGD2 2-fluoropropionyl labeled PEGylated dimeric RGD peptide (PEG3-E[c{RGDyk}]2) uptake, with SUVmax maximum standardized uptake value of 2.4-9.7 (mean, 5.0 ± 2.3) at 60 minutes, compared with (18)F- FDG fluorine 18 fluorodeoxyglucose uptake with SUVmax maximum standardized uptake value of 2.2-14.6 (mean, 6.6 ± 4.2). Data from this pilot phase study suggest that (18)F- FPPRGD2 2-fluoropropionyl labeled PEGylated dimeric RGD peptide (PEG3-E[c{RGDyk}]2) is a safe PET radiopharmaceutical agent. Evaluation of (18)F- FPPRGD2 2-fluoropropionyl labeled PEGylated dimeric RGD peptide (PEG3-E[c{RGDyk}]2) in participants with breast cancer demonstrated significant uptake in the primary lesion and in the metastases. Larger cohorts are required to confirm these preliminary findings.

Dimeric [(68)Ga]DOTA-RGD peptide targeting αvβ 3 integrin reveals extracellular matrix alterations after myocardial infarction.

We evaluated a dimeric RGD-peptide, [(68)Ga]DOTA-E-[c(RGDfK)]2, for positron emission tomography (PET) imaging of myocardial integrin expression associated with extracellular matrix remodeling after myocardial infarction (MI) in rat. Male Sprague-Dawley rats were studied at 7 days and 4 weeks after MI induced by permanent ligation of the left coronary artery and compared with sham-operated controls. In vivo imaging revealed higher tracer uptake in the infarcted area than in the remote non-infarcted myocardium of the same rats both at 7 days (MI/remote ratio, 2.25 ± 0.24) and 4 weeks (MI/remote ratio, 2.13 ± 0.37) post-MI. Compared with sham-operated rats, tracer uptake was higher also in the remote, non-infarcted myocardium of MI rats both at 7 days and 4 weeks where it coincided with an increased interstitial fibrosis. Standardized uptake values correlated well with the results of tracer kinetic modeling. Autoradiography confirmed the imaging results showing 5.1 times higher tracer uptake in the infarcted than remote area. Tracer uptake correlated with the amount of β3 integrin subunits in the infarcted area. Our results show that integrin-targeting [(68)Ga]DOTA-E-[c(RGDfK)]2 is a potential tracer for monitoring of myocardial extracellular matrix remodeling after MI using PET.

Utilization of a novel electrochemical 90Sr/90Y generator for the preparation of 90Y-labeled RGD peptide dimer in clinically relevant dose

Abstract The work reported in this paper provides a systematic study towards the development of an optimized strategy for preparation of a clinically relevant dose of 90Y-labeled dimeric RGD peptide derivative, DOTA-E[c(RGDfK)]2 [DOTA-(RGD)2] for in vivo targeted therapy utilizing 90Y obtained from a novel electrochemical 90Sr/90Y generator. The performance of the generator was evaluated to ensure its suitability for providing 90Y in adequate quantity and purity required for formulation of clinically relevant dose for PRRT. 90Y-DOTA-(RGD)2 was synthesized in high yield (86.2 ± 2.5%) and radiochemical purity (98.4 ± 0.5%) using clinically relevant dose (∼3.8 GBq) of 90Y. In vitro stability studies revealed that the radiolabeled conjugate retained its radiochemical purity in normal saline and human serum. Preliminary biodistribution studies carried out in C57/BL6 mice bearing melanoma tumors showed that the preparation exhibited significant tumor uptake (5.30 ± 0.78% of injected activity at 30 min post-injection) with good tumor to background ratio. The optimized radiolabeling protocol seems to be an attractive strategy which is largely viewed as a springboard to realize scope of developing 90Y labeled cyclic RGD peptides for targeted therapy of tumors over-expressing integrin-α v β 3 receptors.

Anti-tumor effect of integrin targeted (177)Lu-3PRGD2 and combined therapy with Endostar.

Purpose: Targeted radiotherapy (TRT) is an emerging approach for tumor treatment. Previously, 3PRGD2 (a dimeric RGD peptide with 3 PEG4 linkers) has been demonstrated to be of advantage for integrin αvβ3 targeting. Given the promising results of 99mTc-3PRGD2 for lung cancer detection in human beings, we are encouraged to investigate the radiotherapeutic efficacy of radiolabeled 3PRGD2. The goal of this study was to investigate and optimize the integrin αvβ3 mediated therapeutic effect of 177Lu-3PRGD2 in the animal model.Experimental Design: Biodistribution, gamma imaging and maximum tolerated dose (MTD) studies of 177Lu-3PRGD2 were performed. The targeted radiotherapy (TRT) with single dose and repeated doses as well as the combined therapy of TRT and the anti-angiogenic therapy (AAT) with Endostar were conducted in U87MG tumor model. The hematoxylin and eosin (H&E) staining and immunochemistry (IHC) were performed post-treatment to evaluate the therapeutic effect.Results: The U87MG tumor uptake of 177Lu-3PRGD2 was relatively high (6.03 ± 0.65 %ID/g, 4.62 ± 1.44 %ID/g, 3.55 ± 1.08 %ID/g, and 1.22 ± 0.18 %ID/g at 1 h, 4 h, 24 h, and 72 h postinjection, respectively), and the gamma imaging could visualize the tumors clearly. The MTD of 177Lu-3PRGD2 in nude mice (>111 MBq) was twice to that of 90Y-3PRGD2 (55.5 MBq). U87MG tumor growth was significantly delayed by 177Lu-3PRGD2 TRT. Significantly increased anti-tumor effects were observed in the two doses or combined treatment groups.Conclusion: The two-dose TRT and combined therapy with Endostar potently enhanced the tumor growth inhibition, but the former does not need to inject daily for weeks, avoiding a lot of unnecessary inconvenience and suffering for patients, which could potentially be rapidly translated into clinical practice in the future.

Comparison of Three Dimeric 18F-AlF-NOTA-RGD Tracers

RGD peptide-based radiotracers are well established as integrin αvβ3 imaging probes to evaluate tumor angiogenesis or tissue remodeling after ischemia or infarction. In order to optimize the labeling process and pharmacokinetics of the imaging probes, we synthesized three dimeric RGD peptides with or without PEGylation and performed in vivo screening. Radiolabeling was achieved through the reaction of F-18 aluminum-fluoride complex with the cyclic chelator, 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA). Three imaging probes were synthesized as (18)F-AlF-NOTA-E[c(RGDfK)]2, (18)F-AlF-NOTA-PEG4-E[c(RGDfK)]2, and (18)F-AlF-NOTA-E[PEG4-c(RGDfk)]2. The receptor binding affinity was determined by competitive cell binding assay, and the stability was evaluated by mouse serum incubation. Tumor uptake and whole body distribution of the three tracers were compared through direct tissue sampling and PET quantification of U87MG tumor-bearing mice. All three compounds remained intact after 120 min incubation with mouse serum. They all had a rapid and relatively high tracer uptake in U87MG tumors with good target-to-background ratios. Compared with the other two tracers, (18)F-AlF-NOTA-E[PEG4-c(RGDfk)]2 had the highest tumor uptake and the lowest accumulation in the liver. The integrin receptor specificity was confirmed by co-injection of unlabeled dimeric RGD peptide. The rapid one-step radiolabeling strategy by the complexation of (18)F-aluminum fluoride with NOTA-peptide conjugates was successfully applied to synthesize three dimeric RGD peptides. Among the three probes developed, (18)F-AlF-NOTA-E[PEG4-c(RGDfk)]2 with relatively low liver uptake and high tumor accumulation appears to be a promising candidate for further translational research.

A novel concept of radiosynthesis of a 99mTc-labeled dimeric RGD peptide as a potential radiotracer for tumor imaging

Radiolabeled Arg-Gly-Asp (RGD) peptides are promising agents for non invasive imaging of αvβ3 expression in malignant tumors. The integrin αvβ3 binding affinity and consequent tumor uptake could be improved when a dimeric RGD peptide is used as the targeting moiety instead of a monomer. Towards this, a novel approach was envisaged to synthesize a 99mTc labeled dimeric RGD derivative using a RGD monomer and [99mTcN]+2 intermediate. The dithiocarbamate derivative of cyclic RGD peptide G3-c(RGDfK) (G3=Gly-Gly-Gly, f=Phe, K=Lys) was synthesized and radiolabeled with [99mTcN]+2 intermediate to form the 99mTcN-[G3-c(RGDfK)]2 complex in high yield (∼98%). Biodistribution studies carried out in C57/BL6 mice bearing melanoma tumors showed good tumor uptake [4.61±0.04% IA/g at 30min post-injection] with fast clearance of the activity from non-target organs/tissue. Scintigraphic imaging studies showed visible accumulation of activity in the tumor with appreciable target to background ratio.

Imaging integrin alpha‐v‐beta‐3 expression in tumors with an 18F‐labeled dimeric RGD peptide

Integrin αv β3 receptors are expressed on activated endothelial cells during neovascularization to maintain tumor growth. Many radiolabeled probes utilize the tight and specific association between the arginine-glycine-aspartatic acid (RGD) peptide and integrin αv β3 , but one main obstacle for any clinical application of these probes is the laborious multistep radiosynthesis of (18)F. In this study, the dimeric RGD peptide, E-[c(RGDfK)]2, was conjugated with NODAGA and radiolabeled with (18)F in a simple one-pot process with a radiolabeling yield of 20%, the whole process lasting only 45 min. NODAGA-E-[c(RGDfK)]2 labeled with (18)F at a specific activity of 1.8 MBq nmol(-1) and a radiochemical purity of 100% could be achieved. The logP value of (18)F-labeled NODAGA-E-[c(RGDfK)]2 was -4.26 ± 0.02. In biodistribution studies, (18)F-NODAGA-E-[c(RGDfK)]2 cleared rapidly from the blood with 0.03 ± 0.01 percentage injected dose per gram (%ID g(-1)) in the blood at 2 h p.i., mainly via the kidneys, and showed good in vivo stability. Tumor uptake of (18)F-NODAGA-E-[c(RGDfK)]2 (3.44 ± 0.20 %ID g(-1), 2 h p.i.) was significantly lower than that of reference compounds (68) Ga-labeled NODAGA-E-[c(RGDfK)]2 (6.26 ± 0.76 %ID g(-1) ; p <0.001) and (111) In-labeled NODAGA-E-[c(RGDfK)]2 (4.99 ± 0.64 %ID g(-1) ; p < 0.01). Co-injection of an excess of unlabeled NODAGA-E-[c(RGDfK)]2 along with (18)F-NODAGA-E-[c(RGDfK)]2 resulted in significantly reduced radioactivity concentrations in the tumor (0.85 ± 0.13 %ID g(-1)). The αv β3 integrin-expressing SK-RC-52 tumor could be successfully visualized by microPET with (18)F-labeled NODAGA-E-[c(RGDfK)]2 . In conclusion, NODAGA-E-[c(RGDfK)]2 could be labeled rapidly with (18)F using a direct aqueous, one-pot method and it accumulated specifically in αv β3 integrin-expressing SK-RC-52 tumors, allowing for visualization by microPET.

Integrin αvβ3-Targeted Radiotracer 99mTc-3P-RGD2 Useful for Noninvasive Monitoring of Breast Tumor Response to Antiangiogenic Linifanib Therapy but not Anti-Integrin αvβ3 RGD2 Therapy

Purpose: 99mTc-3P-RGD2 is a 99mTc-labeled dimeric cyclic RGD peptide that binds to integrin αvβ3 with high affinity and specificity. The purpose of this study was to demonstrate the utility of 99mTc-3P-RGD2 SPECT/CT (single photon emission computed tomography/computed tomography) as a molecular imaging tool for noninvasive monitoring breast tumor early response to antiangiogenesis therapy with linifanib, and to illustrate its limitations in monitoring the efficacy of anti-αvβ3 treatment.Methods: To support SPECT/CT imaging, biodistribution and therapy studies, the xenografted breast cancer model was established by subcutaneous injection of 5 × 106 MDA-MB-435 cells into the fat pad of each athymic nude mouse. Linifanib (ABT-869) was used as antiangiogenesis agent. The tumor volume was 180 ± 90 mm3 on the day (-1 day) before baseline SPECT/CT. Each animal was treated twice daily with vehicle or 12.5 mg/kg linifanib. Longitudinal 99mTc-3P-RGD2 SPECT/CT imaging was performed on days -1, 1, 4 and 11. Tumors were harvested at each time point for pathological analysis of hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Tumor uptake of 99mTc-3P-RGD2 was calculated from SPECT/CT quantification. When cyclic peptide E[c(RGDfK)]2 (RGD2) was used as the anti-αvβ3 agent, SPECT/CT images were obtained only at 7 and 21 days after last RGD2 dose.Results: The tumor uptake of 99mTc-3P-RGD2 from SPECT/CT quantification was almost identical to that from biodistribution. There was a dramatic reduction in both %ID and %ID/cm3 tumor uptake of 99mTc-3P-RGD2 during the first 24 hours of linifanib therapy. The therapeutic effect of linifanib was on both tumor cells and vasculature, as determined by IHC analysis of integrin αvβ3 and CD31. Changes in tumor vasculature were further confirmed by pathological H&E analysis of tumor tissues. While its %ID tumor uptake increased steadily in vehicle-treated group, the %ID tumor uptake of 99mTc-3P-RGD2 decreased in linifanib-treated group slowly over the 11-day study period. The degree of tumor response to linifanib therapy correlated well to the integrin αvβ3 expression levels before linifanib therapy.Conclusion: 99mTc-3P-RGD2 is an excellent radiotracer for monitoring integrin αvβ3 expression during and after linifanib therapy. 99mTc-3P-RGD2 SPECT/CT is an useful molecular imaging tool for patient selection before antiangiogenic and anti-αvβ3 therapy; but it would be difficult to use 99mTc-3P-RGD2 for accurate and noninvasive monitoring of early tumor response to anti-αvβ3 therapy.