<Superscript>68</Superscript>Ga-labeled multimeric RGD peptides for microPET imaging of integrin α<Subscript>v</Subscript>β<Subscript>3</Subscript> expression

PURPOSE: We and others have reported that (18)F- and (64)Cu-labeled arginine-glycine-aspartate (RGD) peptides allow positron emission tomography (PET) quantification of integrin alpha(v)beta(3) expression in vivo. However, clinical translation of these radiotracers is partially hindered by the necessity of cyclotron facility to produce the PET isotopes. Generator-based PET isotope (68)Ga, with a half-life of 68 min and 89% positron emission, deserves special attention because of its independence of an onsite cyclotron. The goal of this study was to investigate the feasibility of (68)Ga-labeled RGD peptides for tumor imaging. METHODS: Three cyclic RGD peptides, c(RGDyK) (RGD1), E[c(RGDyK)](2) (RGD2), and E{E[c(RGDyK)](2)}(2) (RGD4), were conjugated with macrocyclic chelator 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and labeled with (68)Ga. Integrin affinity and specificity of the peptide conjugates were assessed by cell-based receptor binding assay, and the tumor targeting efficacy of (68)Ga-labeled RGD peptides was evaluated in a subcutaneous U87MG glioblastoma xenograft model. RESULTS: U87MG cell-based receptor binding assay using (125)I-echistatin as radioligand showed that integrin affinity followed the order of NOTA-RGD4 > NOTA-RGD2 > NOTA-RGD1. All three NOTA conjugates allowed nearly quantitative (68)Ga-labeling within 10 min (12-17 MBq/nmol). Quantitative microPET imaging studies showed that (68)Ga-NOTA-RGD4 had the highest tumor uptake but also prominent activity accumulation in the kidneys. (68)Ga-NOTA-RGD2 had higher tumor uptake (e.g., 2.8 +/- 0.1%ID/g at 1 h postinjection) and similar pharmacokinetics (4.4 +/- 0.4 tumor/muscle ratio, 2.0 +/- 0.1 tumor/liver ratio, and 1.1 +/- 0.1 tumor/kidney ratio) compared with (68)Ga-NOTA-RGD1. CONCLUSIONS: The dimeric RGD peptide tracer (68)Ga-NOTA-RGD2 with good tumor uptake and favorable pharmacokinetics warrants further investigation for potential clinical translation to image integrin alpha(v)beta(3).     

99Tcm标记RGD环肽四聚体在神经胶质瘤裸鼠模型中的显像研究

目的 制备99Tcm标记的含有精氨酸-甘氨酸-天冬氨酸(Arg-Gly-Asp,RGD)序列的环肽四聚体99Tcm-联肼尼克酰胺(HYNIC)-E{E[c(RGDfK)]2}2,评价其在整合素αvβ3表达阳性的荷人神经胶质瘤裸鼠模型的生物分布和显像.方法 以HYNIC为双功能螫合剂,以三羟甲基甘氨酸(tricine)和三苯基膦三磺酸钠(TPfffS)为协同配体,采用两步法制备99Tcm-HYNIC-E{E[c(RGDfK)2}2.通过体外受体竞争结合实验比较e(RGDyK)单体、HYNIC-E[c(RGDfK)2二聚体和HYNIC-E{E[c(RGDfK)]2}2四聚体与整合素αvβ3亲和力.生物分布实验数据显示,99Tcm-HYNIC-E{E[c(RGDtK)]2}2主要经肾排泄;注射后1h,肿瘤对99Tcm-HYNIC-E{E[c(RGDfK)]2}2的摄取为99Tcm-HYNIC-E[c(RG-DfK)]2的2倍,分别为(10.32±0．07)％ID／g和(5.15±O.52)％ID／g,与体外受体竞争结合实验数据相一致;注射后4h,肿瘤对99Tcm-HYNIC-E{E[c(RGDfK)]2}2的摄取仍达(9.35.4±1.35)％ID／g,表明标记物在肿瘤中的滞留时间足够长.r显像结果显示,注射后1h肿瘤清晰可见.注射后4h显像效果更佳.结论 99Tcm-HYNIC-E{E[c(RGDfK)]2}2具有较高的肿瘤摄取和较长的肿瘤滞留时间,可以用于整合素αvβ3表达阳性肿瘤的显像;放射性核素(如90Y)标记的RGD环肽四聚体可用于整合素(αvβ3表达阳性肿瘤的治疗.     

Pegylated Arg-Gly-Asp peptide: 64Cu labeling and PET imaging of brain tumor alphavbeta3-integrin expression.

The alphav-integrins, cell adhesion molecules that are highly expressed on activated endothelial cells and tumor cells but not on dormant endothelial cells or normal cells, present an attractive target for tumor imaging and therapy. We previously coupled a cyclic Arg-Gly-Asp (RGD) peptide, c(RGDyK), with 1,4,7,10-tetraazacyclododecane-N,N',N',N'-tetraacetic acid (DOTA) and labeled the RGD-DOTA conjugate with 64Cu (half-life, 12.8 h; 19% beta+) for solid tumor targeting, with high tumor-to-background contrast. The rapid tumor washout rate and persistent liver and kidney retention of this tracer prompted us to optimize the tracer for improved pharmacokinetic behavior. In this study, we introduced a polyethylene glycol (PEG; molecular weight, 3,400) moiety between DOTA and RGD and evaluated the 64Cu-DOTA-PEG-RGD tracer for microPET imaging in brain tumor models. METHODS: DOTA was activated in situ and conjugated with RGD-PEG-NH2 under slightly basic conditions. alphavbeta3-Integrin-binding affinity was evaluated with a solid-phase receptor-binding assay in the presence of 125I-echistatin. Female nude mice bearing subcutaneous U87MG glioblastoma xenografts were administered 64Cu-DOTA-PEG-RGD, and the biodistributions of the radiotracer were evaluated from 30 min to 4 h after injection. microPET (20 min of static imaging at 1 h after injection) and then quantitative autoradiography were used for tumor visualization and quantification. The same tracer was also applied to an orthotopic U87MG model for tumor detection. RESULTS: The radiotracer was synthesized with a high specific activity (14,800-29,600 GBq/mmol [400-800 Ci/mmol]). The c(RGDyK)-PEG-DOTA ligand showed intermediate binding affinity for alphavbeta3-integrin (50% inhibitory concentration, 67.5 +/- 7.8 nmol/L [mean +/- SD]). The pegylated RGD peptide demonstrated rapid blood clearance (0.57 +/- 0.15 percentage injected dose [%ID]/g [mean +/- SD] at 30 min after injection and 0.03 +/- 0.02 %ID/g at 4 h after injection). Activity accumulation in the tumor was rapid and high at early time points (2.74 +/- 0.45 %ID/g at 30 min after injection), and some activity washout was seen over time (1.62 +/- 0.18 %ID/g at 4 h after injection). Compared with (64)Cu-DOTA-RGD, this tracer showed improved in vivo kinetics, with significantly reduced liver uptake (0.99 +/- 0.08 %ID/g vs. 1.73 +/- 0.39 %ID/g at 30 min after injection and 0.58 +/- 0.07 %ID/g vs. 2.57 +/- 0.49 %ID/g at 4 h after injection). The pegylated RGD peptide showed higher renal accumulation at early time points (3.51 +/- 0.24 %ID/g vs. 2.18 +/- 0.23 %ID/g at 30 min after infection) but more rapid clearance (1.82 +/- 0.29 %ID/g vs. 2.01 +/- 0.25 %ID/g at 1 h after injection) than 64Cu-DOTA-RGD. The integrin receptor specificity of this radiotracer was demonstrated by blocking of tumor uptake by coinjection with nonradiolabeled c(RGDyK). The high tumor-to-organ ratios for the pegylated RGD peptide tracer (at 1 h after injection: tumor-to-blood ratio, 20; tumor-to-muscle ratio, 12; tumor-to-liver ratio, 2.7; and tumor-to-kidney ratio, 1.2) were confirmed by microPET and autoradiographic imaging in a subcutaneous U87MG tumor model. This tracer was also able to detect an orthotopic brain tumor in a model in which U87MG cells were implanted into the mouse forebrain. Although the magnitude of tumor uptake in the orthotopic xenograft was lower than that in the subcutaneous xenograft, the orthotopic tumor was still visualized with clear contrast from normal brain tissue. CONCLUSION: This study demonstrated the suitability of a PEG moiety for improving the in vivo kinetics of a 64Cu-RGD peptide tracer without compromising the tumor-targeting ability and specificity of the peptide. Systematic investigations of the effects of the size and geometry of PEG on tumor targeting and in vivo kinetics will lead to the development of radiotracers suitable for clinical applications such as visualizing and quantifying alphav-integrin expression by PET. In addition, the same ligand labeled with therapeutic radionuclides may be applicable for integrin-targeted internal radiotherapy.     

microPET of tumor integrin alphavbeta3 expression using 18F-labeled PEGylated tetrameric RGD peptide (18F-FPRGD4).

In vivo imaging of alpha(v)beta(3) expression has important diagnostic and therapeutic applications. Multimeric cyclic RGD peptides are capable of improving the integrin alpha(v)beta(3)-binding affinity due to the polyvalency effect. Here we report an example of (18)F-labeled tetrameric RGD peptide for PET of alpha(v)beta(3) expression in both xenograft and spontaneous tumor models. METHODS: The tetrameric RGD peptide E{E[c(RGDyK)](2)}(2) was derived with amino-3,6,9-trioxaundecanoic acid (mini-PEG; PEG is poly(ethylene glycol)) linker through the glutamate alpha-amino group. NH(2)-mini-PEG-E{E[c(RGDyK)](2)}(2) (PRGD4) was labeled with (18)F via the N-succinimidyl-4-(18)F-fluorobenzoate ((18)F-SFB) prosthetic group. The receptor-binding characteristics of the tetrameric RGD peptide tracer (18)F-FPRGD4 were evaluated in vitro by a cell-binding assay and in vivo by quantitative microPET imaging studies. RESULTS: The decay-corrected radiochemical yield for (18)F-FPRGD4 was about 15%, with a total reaction time of 180 min starting from (18)F-F(-). The PEGylation had minimal effect on integrin-binding affinity of the RGD peptide. (18)F-FPRGD4 has significantly higher tumor uptake compared with monomeric and dimeric RGD peptide tracer analogs. The receptor specificity of (18)F-FPRGD4 in vivo was confirmed by effective blocking of the uptake in both tumors and normal organs or tissues with excess c(RGDyK). CONCLUSION: The tetrameric RGD peptide tracer (18)F-FPRGD4 possessing high integrin-binding affinity and favorable biokinetics is a promising tracer for PET of integrin alpha(v)beta(3) expression in cancer and other angiogenesis related diseases.     

(64)Cu-labeled tetrameric and octameric RGD peptides for small-animal PET of tumor alpha(v)beta(3) integrin expression.

Integrin alpha(v)beta(3) plays a critical role in tumor angiogenesis and metastasis. Suitably radiolabeled cyclic arginine-glycine-aspartic (RGD) peptides can be used for noninvasive imaging of alpha(v)beta(3) expression and targeted radionuclide therapy. In this study, we developed (64)Cu-labeled multimeric RGD peptides, E{E[c(RGDyK)](2)}(2) (RGD tetramer) and E(E{E[c(RGDyK)](2)}(2))(2) (RGD octamer), for PET imaging of tumor integrin alpha(v)beta(3) expression. METHODS: Both RGD tetramer and RGD octamer were synthesized with glutamate as the linker. After conjugation with 1,4,7,10-tetra-azacyclododecane-N,N',N',N'-tetraacetic acid (DOTA), the peptides were labeled with (64)Cu for biodistribution and small-animal PET imaging studies (U87MG human glioblastoma xenograft model and c-neu oncomouse model). A cell adhesion assay, a cell-binding assay, receptor blocking experiments, and immunohistochemistry were also performed to evaluate the alpha(v)beta(3)-binding affinity/specificity of the RGD peptide-based conjugates in vitro and in vivo. RESULTS: RGD octamer had significantly higher integrin alpha(v)beta(3)-binding affinity and specificity than RGD tetramer analog (inhibitory concentration of 50% was 10 nM for octamer vs. 35 nM for tetramer). (64)Cu-DOTA-RGD octamer had higher tumor uptake and longer tumor retention than (64)Cu-DOTA-RGD tetramer in both tumor models tested. The integrin alpha(v)beta(3) specificity of both tracers was confirmed by successful receptor-blocking experiments. The high uptake and slow clearance of (64)Cu-DOTA-RGD octamer in the kidneys was attributed mainly to the integrin positivity of the kidneys, significantly higher integrin alpha(v)beta(3)-binding affinity, and the larger molecular size of the octamer, as compared with the other RGD analogs. CONCLUSION: Polyvalency has a profound effect on the receptor-binding affinity and in vivo kinetics of radiolabeled RGD multimers. The information obtained here may guide the future development of RGD peptide-based imaging and internal radiotherapeutic agents targeting integrin alpha(v)beta(3).     

Effects of linker variation on the in vitro and in vivo characteristics of an 111In-labeled RGD peptide

INTRODUCTION: Due to the selective expression of the alpha(v)beta3 integrin in tumors, radiolabeled arginine-glycine-aspartic acid (RGD) peptides are attractive candidates for tumor targeting. Minor modifications of these peptides could have a major impact on in vivo characteristics. In this study, we systematically investigated the effects of linker modification between two cyclic RGD sequences and DOTA (1,4,7,10-tetraazadodecane-N,N',N",N'-tetraacetic acid) on the in vitro and in vivo characteristics of the tracer. METHODS: A dimeric RGD peptide was synthesized and conjugated either directly with DOTA or via different linkers: PEG4 (polyethylene glycol), glutamic acid or lysine. The RGD peptides were radiolabeled with 111In, and their in vitro and in vivo alpha(v)beta3-binding characteristics were determined. RESULTS: LogP values varied between -2.82+/-0.06 and -3.95+/-0.33. The IC50 values for DOTA-E-[c(RGDfK)]2, DOTA-PEG4-E-[c(RGDfK)]2, DOTA-E-E-[c(RGDfK)]2 and DOTA-K-E-[c(RGDfK)]2 were comparable. Two hours after injection, the tumor uptakes of the 111In-labeled compounds were not significantly different. The kidney accumulation of [111In]-DOTA-K-E-[c(RGDfK)]2 [4.05+/-0.20% of the injected dose per gram (ID/g)] was significantly higher as compared with that of [111In]-DOTA-E-[c(RGDfK)]2 (2.63+/-0.19% ID/g; P<.05) as well as that of [111In]-DOTA-E-E-[c(RGDfK)]2 (2.16+/-0.21% ID/g; P<.01). The liver uptake of [111In]-DOTA-E-E-[c(RGDfK)]2 (2.12+/-0.09% ID/g) was significantly higher as compared with that of [111In]-DOTA-E-[c(RGDfK)]2 (1.64+/-0.1% ID/g; P<.05) as well as that of [111In]-DOTA-K-E-[c(RGDfK)]2 (1.52+/-0.04% ID/g; P<.01). CONCLUSIONS: Linker variation did not affect affinity for alpha(v)beta3 and tumor uptake. Insertion of lysine caused enhanced kidney retention; that of glutamic acid also resulted in enhanced retention in the kidneys. PEG4 appeared to be the most suitable linker as compared with glutamic acid and lysine because it has the highest tumor-to-blood ratio and the lowest uptake in the kidney and liver.     

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.     

Improved targeting of the α<Subscript>v</Subscript>β<Subscript>3</Subscript> integrin by multimerisation of RGD peptides

Purpose The integrin α_vβ₃ is expressed on sprouting endothelial cells and on various tumour cell types. Due to the restricted expression of α_vβ₃ in tumours, α_vβ₃ is considered a suitable receptor for tumour targeting. In this study the α_vβ₃ binding characteristics of an ¹¹¹In-labelled monomeric, dimeric and tetrameric RGD analogue were compared. Methods A monomeric (E-c(RGDfK)), dimeric (E-[c(RGDfK)]₂), and tetrameric (E{E[c(RGDfK)]₂}₂) RGD peptide were synthesised, conjugated with DOTA and radiolabelled with ¹¹¹In. In vitro α_vβ₃ binding characteristics were determined in a competitive binding assay. In vivo α_vβ₃ targeting characteristics of the compounds were assessed in mice with SK-RC-52 xenografts. Results The IC₅₀ values for DOTA-E-c(RGDfK), DOTA-E-[c(RGDfK)]₂, and DOTA-E{E[c(RGDfK)]₂}₂were 120 nM, 69.9 nM and 19.6 nM, respectively. At all time points, the tumour uptake of the dimer was significantly higher as compared to that of the monomer. At 8 h p.i., tumour uptake of the tetramer (7.40±1.12%ID/g) was significantly higher than that of the monomer (2.30±0.34%ID/g), p<0.001, and the dimer (5.17±1.22%ID/g), p<0.05. At 24 h p.i., the tumour uptake was significantly higher for the tetramer (6.82±1.41%ID/g) than for the dimer (4.22±0.96%ID/g), p<0.01, and the monomer (1.90±0.29%ID/g), p<0.001. Conclusion Multimerisation of c(RGDfK) resulted in enhanced affinity for α_vβ₃ as determined in vitro. Tumour uptake of a tetrameric RGD peptide was significantly higher than that of the monomeric and dimeric analogues, presumably owing to the enhanced statistical likelihood for rebinding to α_vβ₃.     

Radiolabeling, biodistribution, and dosimetry of (123)I-mAb 14C5: a new mAb for radioimmunodetection of tumor growth and metastasis in vivo.

This study reports on the in vitro evaluation, biodistribution, and dosimetry of (123)I-labeled monoclonal antibody (mAb) 14C5, a new antibody-based agent proposed for radioimmunodetection of tumor growth and metastasis in vivo. METHODS: (123)I-mAb 14C5 was prepared by direct iodination and tested for stability in vitro. Binding assays were performed on human SK-BR-3 and HeLa carcinoma cells to investigate the antigen expression, antibody affinity, and kinetics of tracer binding. For the biodistribution and dosimetry study, 3- to 4-wk-old NMRI mice were injected intravenously with (123)I-mAb 14C5 (148.0 +/- 7.4 kBq per mouse) and killed at preset time intervals. Organs, blood, urine, and feces were counted for radioactivity uptake, and the data were expressed as the percentage injected dose per gram tissue (%ID/g tissue) or %ID. The MIRDOSE3.0 program was applied to extrapolate the estimated absorbed radiation doses for various organs to the human reference adult. RESULTS: (123)I-mAb 14C5 was obtained in radiochemical yields of 85.0% +/- 2.5% and radiochemical purities were >97%. The iodinated antibody demonstrated good in vitro stability with 93.6% +/- 0.1% of (123)I-mAb 14C5 remaining intact at 24 h after radiolabeling. (123)I-mAb 14C5 bound to SK-BR-3 cells (dissociation constant [K(d)] approximately 0.85 +/- 0.17 nmol/L) and HeLa cells (K(d) approximately 1.71 +/- 0.17 nmol/L) with nanomolar affinity and high specificity, whereas both cell types exhibited a high CA14C5 antigen expression (maximum number of binding sites [B(max)] = 40.6 +/- 5.2 and 57.1 +/- 9.6 pmol/L, respectively). In mice, (123)I-mAb 14C5 accumulated primarily in lungs (20.4 %ID/g), liver (15.1 %ID/g), and kidneys (11.1 %ID/g) within 5 min after injection. A delayed uptake was observed in stomach (12.8 %ID/g) and urinary bladder (8.7 %ID/g) at 3 and 6 h, respectively, after injection. Radioactivity clearance was predominantly urinary, with 44.9 +/- 4.5 %ID excreted during the initial 48 h after administration (cumulative amount). The highest absorbed radiation doses determined for the human reference adult were received by the urinary bladder wall (0.1200-0.1210 mGy/MBq), liver (0.0137-0.0274 mGy/MBq), uterus (0.0196-0.0207 mGy/MBq), and lower large intestine wall (0.0139-0.0258 mGy/MBq). The average effective dose resulting from a single (123)I-mAb 14C5 injection was estimated to be 0.017-0.022 mSv/MBq. CONCLUSION: (123)I-mAb 14C5 shows good in vitro biologic activity and favorable biodistribution properties for imaging carcinomas of different origin and provides an acceptable radiation dose to the patient.     

Favorable biokinetic and tumor-targeting properties of 99mTc-labeled glucosamino RGD and effect of paclitaxel therapy.

Compared with the recent advancements in radiohalogenated Arg-Gly-Asp (RGD) peptides for alpha(v)beta(3)-targeted imaging, there has been limited success with (99m)Tc-labeled RGD compounds. In this article, we describe the favorable in vivo kinetics and tumor-imaging properties of a novel (99m)Tc-RGD compound that contains a glucosamine moiety. METHODS: Glucosamino (99m)Tc-d-c(RGDfK) was prepared by incorporating (99m)Tc(CO)(3) to the glucosamino peptide precursor in high radiochemical yield. Cell-binding characteristics were tested on human endothelial cells. Mice bearing RR1022 fibrosarcoma and Lewis lung carcinoma (LLC) tumors were used for in vivo biodistribution and blocking experiments and for imaging studies. Separate LLC-bearing mice underwent antiangiogenic therapy with 0, 20, or 40 mg of paclitaxel per kilogram of body weight every 2 d. Tumor volume was serially monitored, and tumor glucosamino (99m)Tc-d-c(RGDfK) uptake and Western blots of alpha(v) integrin expression were analyzed at day 14. RESULTS: Glucosamino (99m)Tc-d-c(RGDfK) binding to endothelial cells was dose-dependently inhibited by excess RGD. Biodistribution in mice showed rapid blood clearance of glucosamino (99m)Tc-d-c(RGDfK), with substantially lower liver uptake and higher tumor uptake compared with (125)I-c(RGD(I)yV). Tumor uptake was 1.03 +/- 0.21 and 1.18 +/- 0.26 %ID/g at 1 h and 0.85 +/- 0.05 and 0.89 +/- 0.28 %ID/g at 4 h for sarcomas and carcinomas, respectively. Excess RGD blocked uptake by 76.5% and 70.2% for the respective tumors. gamma-Camera imaging allowed clear tumor visualization, with an increase of sarcoma-to-thigh count ratios from 5.5 +/- 0.7 at 1 h to 10.1 +/- 2.2 at 4 h and sustained carcinoma-to-thigh count ratios from 4 to 17 h. Pretreatment with excess cRGDyV significantly reduced tumor contrast on images. Paclitaxel therapy in LLC tumor-bearing mice significantly retarded tumor growth. This was accompanied by a corresponding reduction of tumor glucosamino (99m)Tc-d-c(RGDfK) uptake, which correlated significantly with tumor alpha(v) integrin expression levels. CONCLUSION: Glucosamino (99m)Tc-d-c(RGDfK) has favorable in vivo biokinetics and tumor-imaging properties and may be useful for noninvasive evaluation of tumor integrin expression and response to antiangiogenic therapeutics. Because of the wide accessibility of gamma-cameras and high availability and excellent imaging characteristics of (99m)Tc, glucosamino (99m)Tc-d-c(RGDfK) may be an attractive alternative to radiohalogenated RGD peptides for angiogenesis-imaging research.     

PET imaging of acute and chronic inflammation in living mice

Purpose In this study, we evaluated the 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced acute and chronic inflammation in living mice by PET imaging of TNF-α and integrin α_vβ₃ expression. Methods TPA was topically applied to the right ear of BALB/c mice every other day to create the inflammation model. ⁶⁴Cu-DOTA-etanercept and ⁶⁴Cu-DOTA-E{E[c(RGDyK)]₂}₂ were used for PET imaging of TNF-α and integrin α_vβ₃ expression in both acute and chronic inflammation. Hematoxylin and eosin staining, ex vivo autoradiography, direct tissue sampling, and immunofluorescence staining were also performed to confirm the non-invasive PET imaging results. Results The ear thickness increased significantly and the TNF-α level more than tripled after a single TPA challenge. MicroPET imaging using ⁶⁴Cu-DOTA-etanercept revealed high activity accumulation in the inflamed ear, reaching 11.1 ± 1.3, 13.0 ± 2.0, 10.9 ± 1.4, 10.2 ± 2.2%ID/g at 1, 4, 16, and 24 h post injection, respectively (n = 3). Repeated TPA challenges caused TPA-specific chronic inflammation and reduced ⁶⁴Cu-DOTA-etanercept uptake due to lowered TNF-α expression. ⁶⁴Cu-DOTA-E{E[c(RGDyK)]₂}₂ uptake in the chronically inflamed ears (after four and eight TPA challenges) was significantly higher than in the control ears and those after one TPA challenge. Immunofluorescence staining revealed increased integrin β₃ expression, consistent with the non-invasive PET imaging results using ⁶⁴Cu-DOTA-E{E[c(RGDyK)]₂}₂ as an integrin α_vβ₃-specific radiotracer. Biodistribution and autoradiography studies further confirmed the quantification capability of microPET imaging. Conclusion Successful PET imaging of TNF-α expression in acute inflammation and integrin α_vβ₃ expression in chronic inflammation provides the rationale for multiple target evaluation over time to fully understand the inflammation processes. Qizhen Cao and Weibo Cai contributed equally to this work.     

Quantitative PET imaging of tumor integrin alphavbeta3 expression with 18F-FRGD2.

The development of noninvasive methods to visualize and quantify integrin alpha(v)beta(3) expression in vivo appears to be crucial for the success of antiangiogenic therapy based on integrin antagonism. Precise documentation of integrin receptor levels will allow appropriate selection of patients who will most likely benefit from an antiintegrin treatment regimen. Imaging can also be used to provide an optimal dosage and time course for treatment based on receptor occupancy studies. In addition, imaging integrin expression will be important to evaluate antiintegrin treatment efficacy and to develop new therapeutic drugs with favorable tumor targeting and in vivo kinetics. We labeled the dimeric RGD peptide E[c(RGDyK)](2) with (18)F and evaluated its tumor-targeting efficacy and pharmacokinetics of (18)F-FB-E[c(RGDyK)](2) ((18)F-FRGD2). METHODS: E[c(RGDyK)](2) was labeled with (18)F by conjugation coupling with N-succinimidyl-4-(18)F-fluorobenzoate ((18)F-SFB) under a slightly basic condition. The in vivo metabolic stability of (18)F-FRGD2 was determined. The diagnostic value after injection of (18)F-FRGD2 was evaluated in various xenograft models by dynamic microPET followed by ex vivo quantification of tumor integrin level. RESULTS: Starting with (18)F(-) Kryptofix 2.2.2./K(2)CO(3) solution, the total reaction time for (18)F-FRGD2, including final high-performance liquid chromatography purification, is about 200 +/- 20 min. Typical decay-corrected radiochemical yield is 23% +/- 2% (n = 20). (18)F-FRGD2 is metabolically stable. The binding potential extrapolated from graphical analysis of PET data and Logan plot correlates well with the receptor density measured by sodium dodecyl sulfate polyacrylamide electrophoresis and autoradiography in various xenograft models. The tumor-to-background ratio at 1 h after injection of (18)F-FRGD2 also gives a good linear relationship with the tumor tissue integrin level. CONCLUSION: The dimeric RGD peptide tracer (18)F-FRGD2, with high integrin specificity and favorable excretion profile, may be translated into the clinic for imaging integrin alpha(v)beta(3) expression. The binding potential calculated from simplified tracer kinetic modeling such as the Logan plot appears to be an excellent indicator of tumor integrin density.     

Radiolabeled RGD uptake and alphav integrin expression is enhanced in ischemic murine hindlimbs.

Radiolabeled RGD peptides that target alpha(v)beta3 integrin are promising tracers for imaging tumor angiogenesis. Integrins and angiogenesis also play important roles in healing of ischemic lesions. Thus, we investigated the biodistribution of radiolabeled RGD and expression of alpha(v) integrin in a mouse model of hindlimb ischemia. METHODS: 125I-3-Iodo-D-Tyr4-cyclo(-Arg-Gly-Asp-D-Tyr-Val-) (125I-c(RGD(I)yV)) was synthesized and tested for endothelial binding. Hindlimb ischemia was induced in ICR mice through femoral artery ablation, and perfusion was measured with laser Doppler blood flowmetry. 125I-c(RGD(I)yV) biodistribution was evaluated in control animals (n = 7) and ischemic models on day 3, 8, or 14 (n = 6 each). Control experiments were performed using a radiolabeled peptide with a scrambled amino acid sequence (125I-GfVGV). Microsections of hindlimb tissue were immunostained for alpha(v) integrin expression and stained with alkaline phosphatase to localize vascular endothelial cells. RESULTS: 125I-c(RGD(I)yV) retained specific binding to human umbilical vein endothelial cells. Perfusion in ischemic hindlimbs immediately fell to 10% +/- 4% of contralateral levels and gradually recovered to 22% +/- 11% and 64% +/- 9% on days 8 and 14, respectively. 125I-c(RGD(I)yV) uptake in ischemic muscles significantly increased from a control level of 0.16 +/- 0.05 %ID/g (percentage injected dose per gram of tissue) to 0.85 +/- 0.76 %ID/g at day 3, 0.43 +/- 0.23 %ID/g at day 8, and 0.43 +/- 0.28 %ID/g at day 14 (all P < 0.05). Ischemic muscle-to-lung count ratios had a virtually identical trend: 0.42 +/- 0.25 for controls, 2.34 +/- 1.70 at day 3 (P < 0.02), 1.46 +/- 0.52 at day 8 (P < 0.001), and 1.39 +/- 0.94 at day 14 (P < 0.02). In contrast, uptake of the control peptide in ischemic hindlimbs was not different from that of controls. Immunohistochemistry revealed substantially increased alpha(v) integrin staining in ischemic hindlimb tissue. CONCLUSION: Radioiodine RGD uptake is significantly enhanced in ischemic hindlimbs of a mouse model, and is accompanied by an increase in alpha(v) integrin expression. Further investigation is thus warranted to illuminate the potential role of radiolabeled RGD for noninvasive monitoring of peripheral ischemic lesions.     

Comparison of two new angiogenesis PET tracers 68Ga-NODAGA-E[c(RGDyK)]2 and 64Cu-NODAGA-E[c(RGDyK)]2; in vivo imaging studies in human xenograft tumors

IntroductionThe aim of this study was to synthesize and perform a side-by-side comparison of two new tumor-angiogenesis PET tracers ⁶⁸Ga-NODAGA-E[c(RGDyK)]₂ and ⁶⁴Cu-NODAGA-E[c(RGDyK)]₂ in vivo using human xenograft tumors in mice. Human radiation burden was estimated to evaluate potential for future use as clinical PET tracers for imaging of neo-angiogenesis.MethodsA ⁶⁸Ge/⁶⁸Ga generator was used for the synthesis of ⁶⁸Ga-NODAGA-E[c(RGDyK)]₂. ⁶⁸Ga and ⁶⁴Cu labeled NODAGA-E[c(RGDyK)]₂ tracers were administrated in nude mice bearing either human glioblastoma (U87MG) or human neuroendocrine (H727) xenograft tumors. PET/CT scans at 3 time points were used for calculating the tracer uptake in tumors (%ID/g), integrin α_Vβ₃ target specificity was shown by blocking with cold NODAGA-E[c(RGDyK)]₂, and biodistribution in normal organs were also examined. From biodistribution data in mice human radiation-absorbed doses were estimated using OLINDA/EXM software.Results⁶⁸Ga-NODAGA-E[c(RGDyK)]₂ was synthesized with a radiochemical purity of 89%–99% and a specific activity (SA) of 16–153 MBq/nmol. ⁶⁴Cu-NODAGA-E[c(RGDyK)]₂ had a purity of 92%–99% and an SA of 64–78 MBq/nmol.Both tracers showed similar uptake in xenograft tumors 1 h after injection (U87MG: 2.23 vs. 2.31%ID/g; H727: 1.53 vs. 1.48%ID/g). Both RGD dimers showed similar tracer uptake in non-tumoral tissues and a human radiation burden of less than 10 mSv with an administered dose of 200 MBq was estimated.Conclusion⁶⁸Ga-NODAGA-E[c(RGDyK)]₂ and ⁶⁴Cu-NODAGA-E[c(RGDyK)]₂ can be easily synthesized and are both promising candidates for PET imaging of integrin α_Vβ₃ positive tumor cells. ⁶⁸Ga-NODAGA-E[c(RGDyK)]₂ showed slightly more stable tumor retention. With the advantage of in-house commercially ⁶⁸Ge/⁶⁸Ga generators, ⁶⁸Ga-NODAGA-E[c(RGDyK)]₂ may be the best choice for future clinical PET imaging in humans.     

Preparation of a promising angiogenesis PET imaging agent: 68Ga-labeled c(RGDyK)-isothiocyanatobenzyl-1,4,7-triazacyclononane-1,4,7-triacetic acid and feasibility studies in mice.

Arg-Gly-Asp (RGD) derivatives have been labeled with various radioisotopes for the imaging of angiogenesis in ischemic tissue, in which alpha(v)beta(3) integrin plays an important role. In this study, cyclic Arg-Gly-Asp-D-Tyr-Lys [c(RGDyK)] was conjugated with 2-(p-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (SCN-Bz-NOTA) and then labeled with (68)Ga. The labeled RGD so produced was subjected to an in vitro binding assay and in vivo biodistribution and PET studies. METHODS: A mixture of SCN-Bz-NOTA (660 nmol) and c(RGDyK) (600 nmol) in 0.1 M sodium carbonate buffer (pH 9.5) was allowed to react for 20 h at room temperature in the dark for thiourea bond formation. The conjugate obtained was purified by semipreparative high-performance liquid chromatography (HPLC). The purified c(RGDyK)-SCN-Bz-NOTA (NOTA-RGD) was then labeled with (68)Ga from a (68)Ge/(68)Ga generator and purified by semipreparative HPLC. A competitive binding assay for c(RGDyK) and NOTA-RGD was performed with (125)I-c(RGDyK) as a radioligand and alpha(v)beta(3) integrin-coated plates as a solid phase. (68)Ga-NOTA-RGD (0.222 MBq/100 microL) was injected, through a tail vein, into mice with hind limb ischemia and into mice bearing human colon cancer SNU-C4 xenografts. Biodistribution and imaging studies were performed at 1 and 2 h after injection. RESULTS: The labeling of NOTA-RGD with (68)Ga was straightforward. The K(i) values of c(RGDyK) and NOTA-RGD were 1.3 and 1.9 nM, respectively. In the biodistribution study, the mean +/- SD uptake of (68)Ga-NOTA-RGD by ischemic muscles was 1.6+/-0.2 percentage injected dose per gram (%ID/g); this uptake was significantly blocked by cold c(RGDyK) to 0.6+/-0.3 %ID/g (P<0.01). Tumor uptake was 5.1+/-1.0 %ID/g, and the tumor-to-blood ratio was 10.3+/-4.8. Small-animal PET revealed rapid excretion through the urine and high levels of tumor and kidney uptake. CONCLUSION: Stable (68)Ga-NOTA-RGD was obtained in a straightforward manner at a high yield and showed a high affinity for alpha(v)beta(3) integrin, specific uptake by angiogenic muscles, a high level of uptake by tumors, and rapid renal excretion. (68)Ga-NOTA-RGD was found to be a promising radioligand for the imaging of angiogenesis.     

PET-based human dosimetry of 18F-galacto-RGD, a new radiotracer for imaging alpha v beta3 expression.

(18)F-Galacto-RGD is a new tracer for PET imaging of alpha v beta3, a receptor involved in a variety of pathologic processes including angiogenesis and metastasis. Our aim was to study the dosimetry of (18)F-galacto-RGD in humans. METHODS: Eighteen patients with various tumors (musculoskeletal tumors [n = 10], melanoma [n = 5], breast cancer [n = 2], or head and neck cancer [n = 1]) were examined. After injection of 133-200 MBq of (18)F-galacto-RGD, 3 consecutive emission scans from the thorax to the pelvis were acquired at 6.7 +/- 2.9, 35.6 +/- 7.6, and 70.4 +/- 12.2 min after injection. Blood samples (n = 4) for metabolite analysis were taken 10, 30, and 120 min after injection. The OLINDA 1.0 program was used to estimate the absorbed radiation dose. RESULTS: Reversed-phase high-performance liquid chromatography of serum revealed that more than 95% of tracer was intact up to 120 min after injection. (18)F-Galacto-RGD showed rapid clearance from the blood pool and primarily renal excretion. Background activity in lung and muscle tissue was low (percentage injected dose per liter at 71 min after injection, 0.56 +/- 0.15 and 0.69 +/- 0.25, respectively). The calculated effective dose was 18.7 +/- 2.4 microSv/MBq, and the highest absorbed radiation dose was in the bladder wall (0.22 +/- 0.03 mGy/MBq). CONCLUSION: (18)F-Galacto-RGD demonstrates high metabolic stability, a favorable biodistribution, and a low radiation dose. Consequently, this tracer can safely be used for noninvasive imaging of molecular processes involving the alpha v beta3 integrin and for the planning and monitoring of therapeutic approaches targeting alpha v beta3.     

18F-Fluorothymidine radiation dosimetry in human PET imaging studies.

3'-Deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) is a PET imaging agent that shows promise for studying cellular proliferation in human cancers. FLT is a nucleoside analog that enters cells and is phosphorylated by human thymidine kinase 1, but the 3' substitution prevents further incorporation into DNA. We estimated the radiation dosimetry for this tracer from data gathered in patient studies. METHODS: Time-dependent tissue concentrations of radioactivity were determined from blood samples and PET images of 18 patients after intravenous injection of (18)F-FLT. Radiation-absorbed doses were calculated using the MIRD Committee methods, taking into account variations that were based on the distribution of activities observed in the individual patients. Effective dose equivalent (EDE) was calculated using International Commission on Radiological Protection Publication 60 tissue weighting factors for the standard man and woman. RESULTS: For a single bladder voiding at 6 h after (18)F-FLT injection, the (18)F-FLT EDE (mean +/- SD) was 0.028 +/- 0.012 mSv/MBq (103 +/- 43 mrem/mCi) for a standard male patient and 0.033 +/- 0.012 mSv/MBq (121 +/- 43 mrem/mCi) for a standard female patient. The organ that received the highest dose was the bladder (male, 0.179 mGy/MBq [662 mrad/mCi]; female, 0.174 mGy/MBq [646 mrad/mCi]), followed by the liver (male, 0.045 mGy/MBq [167 mrad/mCi]; female, 0.064 mGy/MBq [238 mrad/mCi]), the kidneys (male, 0.035 mGy/MBq [131 mrad/mCi]; female, 0.042 mGy/MBq [155 mrad/mCi]), and the bone marrow (male, 0.024 mGy/MBq [89 mrad/mCi]; female, 0.033 mGy/MBq [122 mrad/mCi]). CONCLUSION: Organ dose estimates for (18)F-FLT are comparable to those associated with other commonly performed nuclear medicine tests, and the potential radiation risks associated with (18)F-FLT PET imaging are within accepted limits.     

Preparation and characterization of 99mTc(CO)3-BPy-RGD complex as alphav beta3 integrin receptor-targeted imaging agent.

The aim of this study is to develop a novel arginine-glycine-aspartic acid (RGD) peptide-containing ligand for (99m)Tc labeling as alpha(v)beta(3) integrin receptor-targeted imaging agent. BPy-RGD conjugate was successfully synthesized by coupling of 5-carboxylate-2,2'-bipyridine and c(RGDyK) peptide through EDC/SNHS in aqueous solution and was characterized by MADLI-TOF-MS (m/z=802.72, C(38)H(48)N(11)O(9)). (99m)Tc(CO)(3)-BPy-RGD was prepared by exchange reaction between [(99m)Tc(H(2)O)(3)(CO)(3)](+) and BPy-RGD. Final product was purified by HPLC and tested for octanol/water partition coefficient. Cell-binding assays of BPy-RGD and unmodified c(RGDyK) were tested in MDA-MB-435 cells ((125)I-echistatin as radioligand). Preliminary biodistribution of the (99m)Tc(I)-labeled radiotracer in orthotopic MDA-MB-435 breast tumor xenograft model was also evaluated. The BPy-RGD conjugate had good integrin-binding affinity (50% inhibitory concentration (IC(50))=92.51+/-22.69 nM), slightly lower than unmodified c(RGDyK) (IC(50)=59.07+/-11.03 nM). The hydrophilic radiotracer also had receptor-mediated activity accumulation in MDA-MB-435 tumor (1.45+/-0.25 percentage of injected dose per gram (%ID/g) at 1.5h postinjection (p.i.)), which is known to be integrin positive. After blocking with c(RGDyK), the tumor uptake was reduced from 0.71+/-0.01%ID/g to 0.33+/-0.18%ID/g at 4h p.i. (99m)Tc(I) tricarbonyl complex of cyclic RGD peptide is a promising strategy for integrin targeting. Further modification of the bipyridine-conjugated RGD peptide by using more potent RGD peptides and fine tuning of the tether group between the RGD moiety and (99m)Tc(CO)(3)(+) core to improve the tumor targeting efficacy and in vivo kinetic profiles is currently in progress.     

99Tcm标记新型RGD环肽在神经胶质瘤动物模型的实验研究

目的 评价引入2个聚乙二醇（PEG4）对精氨酸-甘氨酸-天冬氨酸（RGD）环肽二聚体（Dimer：E[c（RGDfK）]2）体外受体结合亲和力和体内药代动力学特征的影响,以及99Tcm标记2PEG4-Dimer用于整合素αvβ3阳性肿瘤显像的前景.方法 用免疫组织化学实验测定U87MG人神经胶质瘤细胞以及肿瘤组织中整合素αvβ3的表达.通过U87MG细胞受体竞争结合实验测定RGD环肽单体c（RGDyK）、联肼尼克酰胺（HYNIC）-Dimer和HYNIC-2PEG4-Dimer对125I-c（RGDyK）的半数抑制浓度（IC50）.采用无亚锡一步法制备99Tcm-HYNIC-Dimer和99Tcm-HYNIC-2PEG4-Dimer,评价＂TcmHYNIC-2PEG4-Dimer在荷U87MG瘤裸鼠的生物分布并进行γ显像.采用非配对t检验法对实验数据进行分析.结果 U87MG细胞和荷瘤裸鼠肿瘤组织中高表达整合素αvβ3.HYNIC-2PEG4-Dimer比c（RGDyK）和HYNIC-Dimer有更高的整合素αvβ3亲和力（IC50分别是0.8,27和2.4 nmol/L）.99Tcm-HYNIC-Dimer和99Tcm-HYNIC-2PEG4-Dimer的99Tcm标记率均＞95%,经Sep-Pek C18柱纯化后其放化纯＞99%.生物分布实验显示,2种标记物均主要经肾排泄,在注射后2h,肿瘤对99Tcm-HYNIC-2PEG4-Dimer的摄取为99Tcm-HYNIC-Dimer的2.7倍[（5.71±0.96）%ID/g和（2.10±0.50）%ID/g],t=4.80,P＜0.05,与体外受体竞争结合实验数据相一致.γ显像结果显示,注射99Tcm-HYNIC-2PEG4-Dimer后0.5 h肿瘤即清晰可见,随时间延长,体内放射性本底明显减低,显像对比度增高.结论 99Tcm-HYNIC-2PEG4-Dimer有希望用于整合素αvβ3阳性肿瘤显像.     

microPET and autoradiographic imaging of GRP receptor expression with 64Cu-DOTA-[Lys3]bombesin in human prostate adenocarcinoma xenografts.

Overexpression of gastrin-releasing peptide (GRP) receptor (GRPR) in both androgen-dependent (AD) and androgen-independent (AI) human neoplastic prostate tissues provides an attractive target for prostate cancer imaging and therapy. The goal of this study was to develop (64)Cu-radiolabeled GRP analogs for PET imaging of GRPR expression in prostate cancer xenografted mice. METHODS: [Lys(3)]bombesin ([Lys(3)]BBN) was conjugated with 1,4,7,10-tetraazadodecane-N,N',N",N"'-tetraacetic acid (DOTA) and labeled with the positron-emitting isotope (64)Cu (half-life = 12.8 h, 19% beta(+)). Receptor binding of DOTA-[Lys(3)]BBN and internalization of (64)Cu-DOTA-[Lys(3)]BBN by PC-3 prostate cancer cells were measured. Tissue biodistribution, microPET, and whole-body autoradiographic imaging of the radiotracer were also investigated in PC-3 (AI)/CRW22 (AD) prostate cancer tumor models. RESULTS: A competitive receptor- binding assay using (125)I-[Tyr(4)]BBN against PC-3 cells yielded a 50% inhibitory concentration value of 2.2 +/- 0.5 nmol/L for DOTA-[Lys(3)]BBN. Incubation of cells with the (64)Cu-labeled radiotracer showed temperature- and time-dependent internalization. At 37 degrees C, >60% of the tracer was internalized within the first 15 min and uptake remained constant for 2 h. Radiotracer uptake was higher in AI PC-3 tumor (5.62 +/- 0.08 %ID/g at 30 min after injection, where %ID/g is the percentage of injected dose per gram) than in AD CWR22 tumor (1.75 +/- 0.05 %ID/g at 30 min after injection). Significant accumulation of the activity in GRPR-positive pancreas was also observed (10.4 +/- 0.15 %ID/g at 30 min after injection). Coinjection of a blocking dose of [Lys(3)]BBN inhibited the activity accumulation in PC-3 tumor and pancreas but not in CWR22 tumor. microPET and autoradiographic imaging of (64)Cu-DOTA-[Lys(3)]BBN in athymic nude mice bearing subcutaneous PC-3 and CWR22 tumors showed strong tumor-to-background contrast. CONCLUSION: This study demonstrates that PET imaging of (64)Cu-DOTA-[Lys(3)]BBN is able to detect GRPR-positive prostate cancer.