<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).     

microPET imaging of glioma integrin {alpha}v{beta}3 expression using (64)Cu-labeled tetrameric RGD peptide.

Integrin alpha(v)beta(3) plays a critical role in tumor-induced angiogenesis and metastasis and has become a promising diagnostic indicator and therapeutic target for various solid tumors. Radiolabeled RGD peptides that are integrin specific can be used for noninvasive imaging of integrin expression level as well as for integrin-targeted radionuclide therapy. METHODS: In this study we developed a tetrameric RGD peptide tracer (64)Cu-DOTA-E{E[c(RGDfK)](2)}(2) (DOTA is 1,4,7,10-tetraazacyclododecane-N,N',N',N'-tetraacetic acid) for PET imaging of integrin alpha(v)beta(3) expression in female athymic nude mice bearing the subcutaneous UG87MG glioma xenografts. RESULTS: The RGD tetramer showed significantly higher integrin binding affinity than the corresponding monomeric and dimeric RGD analogs, most likely due to a polyvalency effect. The radiolabeled peptide showed rapid blood clearance (0.61 +/- 0.01 %ID/g at 30 min and 0.21 +/- 0.01 %ID/g at 4 h after injection, respectively [%ID/g is percentage injected dose per gram]) and predominantly renal excretion. Tumor uptake was rapid and high, and the tumor washout was slow (9.93 +/- 1.05 %ID/g at 30 min after injection and 4.56 +/- 0.51 %ID/g at 24 h after injection). The metabolic stability of (64)Cu-DOTA-E{E[c(RGDfK)](2)}(2) was determined in mouse blood, urine, and liver and kidney homogenates at different times after tracer injection. The average fractions of intact tracer in these organs at 1 h were approximately 70%, 58%, 51%, and 26%, respectively. Noninvasive microPET studies showed significant tumor uptake and good contrast in the subcutaneous tumor-bearing mice, which agreed well with the biodistribution results. Integrin alpha(v)beta(3) specificity was demonstrated by successful blocking of tumor uptake of (64)Cu-DOTA-E{E[c(RGDfK)](2)}(2) in the presence of excess c(RGDyK) at 1 h after injection. The highest absorbed radiation doses determined for the human reference adult were received by the urinary bladder wall (0.262 mGy/MBq), kidneys (0.0296 mGy/MBq), and liver (0.0242 mGy/MBq). The average effective dose resulting from a single (64)Cu-DOTA-E{E[c(RGDfK)](2)}(2) injection was estimated to be 0.0164 mSv/MBq. CONCLUSION: The high integrin and avidity and favorable biokinetics make (64)Cu-DOTA-E{E[c(RGDfK)](2)}(2) a promising agent for peptide receptor radionuclide imaging and therapy of integrin-positive tumors.     

(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).     

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.     

18F-labeled BBN-RGD heterodimer for prostate cancer imaging.

Both bombesin (BBN) analogs and cyclic RGD peptides have been suitably radiolabeled for prostate cancer imaging. However, the limited expression of gastrin-releasing peptide receptor (GRPR) and integrin alpha(v)beta(3) as well as unfavorable in vivo kinetics limited further applications of these imaging agents. We hypothesize that a peptide ligand recognizing both GRPR and integrin will be advantageous because of its dual-receptor-targeting ability. METHODS: A BBN-RGD heterodimer was synthesized from bombesin(7-14) and c(RGDyK) through a glutamate linker and then labeled with (18)F via the N-succinimidyl-4-(18)F-fluorobenzoate ((18)F-SFB) prosthetic group. The receptor-binding characteristics and tumor-targeting efficacy of (18)F-FB-BBN-RGD were tested in vitro and in vivo. RESULTS: FB-BBN-RGD had comparable integrin alpha(v)beta(3)-binding affinity with c(RGDyK) and comparable GRPR-binding affinity with BBN(7-14). (18)F-FB-BBN-RGD had significantly higher tumor uptake compared with monomeric RGD and monomeric BBN peptide tracer analogs at all time points examined. The PC-3 tumor uptake of (18)F-FB-BBN-RGD was inhibited only partially in the presence of an excess amount of unlabeled BBN(7-14) or c(RGDyK) but was blocked completely in the presence of both BBN(7-14) and c(RGDyK). Compared with (18)F-FB-BBN and (18)F-FB-RGD, (18)F-FB-BBN-RGD also had improved pharmacokinetics, resulting in a significantly higher imaging quality. CONCLUSION: Dual integrin alpha(v)beta(3) and GRPR recognition showed significantly improved tumor-targeting efficacy and pharmacokinetics compared with (18)F-labeled RGD and BBN analogs. The same heterodimeric ligand design may also be applicable to other receptor system combinations and other imaging modalities.     

18F-labeled RGD peptide: initial evaluation for imaging brain tumor angiogenesis

Brain tumors are highly angiogenesis dependent. The cell adhesion receptor integrin alpha(v)beta(3) is overexpressed in glioma and activated endothelial cells and plays an important role in brain tumor growth, spread and angiogenesis. Suitably labeled alpha(v)beta(3)-integrin antagonists may therefore be useful for imaging brain tumor associated angiogenesis. Cyclic RGD peptide c(RGDyK) was labeled with (18)F via N-succinimidyl-4-[(18)F]fluorobenzoate through the side-chain epsilon-amino group of the lysine residue. The radiotracer was evaluated in vivo for its tumor targeting efficacy and pharmacokinetics in subcutaneously implanted U87MG and orthotopically implanted U251T glioblastoma nude mouse models by means of microPET, quantitative autoradiography and direct tissue sampling. The N-4-[(18)F]fluorobenzoyl-RGD ([(18)F]FB-RGD) was produced in less than 2 h with 20-25% decay-corrected yields and specific activity of 230 GBq/micromol at end of synthesis. The tracer showed very rapid blood clearance and both hepatobiliary and renal excretion. Tumor-to-muscle uptake ratio at 30 min was approximately 5 in the subcutaneous U87MG tumor model. MicroPET imaging with the orthotopic U251T brain tumor model revealed very high tumor-to-brain ratio, with virtually no uptake in the normal brain. Successful blocking of tumor uptake of [(18)F]FB-RGD in the presence of excess amount of c(RGDyK) revealed receptor specific activity accumulation. Hence, N-4-[(18)F]fluorobenzoyl labeled cyclic RGD peptide [(18)F]FB-RGD is a potential tracer for imaging alpha(v)beta(3)-integrin positive tumors in brain and other anatomic locations.     

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.     

An improved method of 18F peptide labeling: hydrazone formation with HYNIC-conjugated c(RGDyK)

Radiolabeled alpha(v)beta(3)-integrin antagonists are increasingly investigated as a means of imaging angiogenesis. Several methods of labeling alpha(v)beta(3)-integrin binding peptide with (18)F have been reported recently. In the present study, we devised a straightforward means for labeling Arg-Gly-Asp (RGD) peptide with (18)F via hydrazone formation between c(RGDyK)-hydrazinonicotinic acid (HYNIC) (3) and 4-[(18)F]-fluorobenzaldehyde ([(18)F]4). The resulting reaction mixture was purified by HPLC to give 4'-[(18)F]-fluorobenzylidenehydrazone-6-nicotinamide-c(RGDyK) ([(18)F]5). The conjugation efficiency of 3 and 4 to form [(18)F]5 was 95.2%, and the radiochemical purity of [(18)F]5 after purification was >99%. The specific activity of [(18)F]5 estimated by radio-HPLC was 20.5 GBq/mumol (end of synthesis). Competitive binding assay of c(RGDyK) (1) and 5 was performed using [(125)I]iodo-c(RGDyK) as a radioligand, and K(i) values were found to be 2.8 and 21.7 nM, respectively. For the biodistribution study, the angiogenic mouse model was established by inducing unilateral ischemia on the left hindlimbs of ICR mice after femoral artery ablation. Seven days after inducing ischemia, [(18)F]5 was administered to the mice through the tail vein. Ischemic muscle uptake of [(18)F]5 was significantly higher than that of normal muscle (P<.01). Specific uptake was confirmed by coinjection of 1 with [(18)F]5. Here, we successfully labeled RGD peptide with (18)F via hydrazone formation between 3 and 4, resulting to [(18)F]5. [(18)F]5 was found to have high affinity for alpha(v)beta(3)-integrin and to accumulate specifically in ischemic hindlimb muscle of mice. We suggest that (18)F labeling via formation of hydrazone between HYNIC peptide and [(18)F]4 is a useful method for labeling c(RGDyK), which can be applied for imaging angiogenesis.     

Noninvasive imaging of osteoclasts in parathyroid hormone-induced osteolysis using a 64Cu-labeled RGD peptide.

Bone diseases are often a result of increased numbers of osteoclasts, or bone-resorbing cells. Bone metastases are a significant cause of morbidity in many types of cancer. An imaging agent targeting osteoclasts, which are upregulated in osteolytic lesions, may facilitate earlier follow-up in patients with osteolytic or mixed bone metastases. Osteoclasts express high levels of alpha(v)beta3 integrin, to which peptides containing the Arg-Gly-Asp (RGD) sequence are known to bind. We proposed that radiolabeled RGD peptides could be used to detect osteoclasts in lytic bone lesions. METHODS: The cross-bridged macrocyclic chelator 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (CB-TE2A) was conjugated to c(RGDyK) for radiolabeling with 64Cu (t(1/2), 12.7 h; beta+, 17.4%; E(beta+ max), 656 keV; beta-, 39%; E(beta- max), 573 keV). The in vitro affinity of Cu(II)-CB-TE2A-c(RGDyK) for alpha(v)beta3 and alpha(v)beta5 was evaluated in a heterologous competitive binding assay. Ex vivo uptake was examined in osteoclasts prepared from bone marrow macrophages. As a proof of principle, biodistribution and imaging studies were performed on parathyroid hormone (PTH)-induced osteolysis in the calvarium. RESULTS: Cu-CB-TE2A-c(RGDyK) was shown to have a 30-fold higher affinity for alpha(v)beta3 than for alpha(v)beta5. Osteoclasts were shown to specifically take up (64)Cu-CB-TE2A-c(RGDyK). However, bone marrow macrophages showed only nonspecific uptake. PTH treatment increased calvarial uptake of 64Cu-CB-TE2A-c(RGDyK), compared with uptake in mice receiving a sham treatment. In addition, calvarial uptake correlated linearly with the number of osteoclasts on the bone surface. CONCLUSION: These results suggest that 64Cu-CB-TE2A-c(RGDyK) selectively binds alpha(v)beta3 on osteoclasts and may potentially be used to identify increased numbers of osteoclasts in osteolytic bone diseases such as osteolytic bone metastasis and inflammatory osteolysis.     

A thiol-reactive 18F-labeling agent, N-[2-(4-18F-fluorobenzamido)ethyl]maleimide, and synthesis of RGD peptide-based tracer for PET imaging of alpha v beta 3 integrin expression.

The cell adhesion molecule integrin alpha v beta 3 plays a key role in tumor angiogenesis and metastasis. A series of 18F-labeled RGD peptides have been developed for PET of integrin expression based on primary amine-reactive prosthetic groups. In this study we introduced a new method of labeling RGD peptides through a thiol-reactive synthon, N-[2-(4-18F-fluorobenzamido)ethyl]maleimide (18F-FBEM). METHODS: 18F-FBEM was synthesized by coupling N-succinimidyl 4-18F-fluorobenzoate (18F-SFB) with N-(2-aminoethyl)maleimide. After high-pressure liquid chromatography purification, it was allowed to react with thiolated RGD peptides, and the resulting tracers were subjected to receptor-binding assay, in vivo metabolic stability assessment, biodistribution, and microPET studies in murine xenograft models. RESULTS: Conjugation of monomeric and dimeric sulfhydryl-RGD peptides with 18F-FBEM was achieved in high yields (85% +/- 5% nondecay-corrected on the basis of 18F-FBEM). The radiochemical purity of the 18F-labeled peptides was >98% and the specific activity was 100 approximately 150 TBq/mmol. Noninvasive microPET and direct tissue sampling experiments demonstrated that both 18F-FBEM-SRGD (RGD monomer) and 18F-FBEM-SRGD2 (RGD dimer) had integrin-specific tumor uptake in subcutaneous U87MG glioma and orthotopic MDA-MB-435 breast cancer xenografts. CONCLUSION: The new tracer 18F-FBEM-SRGD2 was synthesized with high specific activity via 18F-FBEM and the tracer exhibited high receptor-binding affinity, tumor-targeting efficacy, metabolic stability, as well as favorable in vivo pharmacokinetics. The new synthon 18F-FBEM developed in this study will also be useful for radiolabeling of other thiolated biomolecules.     

Imaging of delayed-type hypersensitivity reaction by PET and 18F-galacto-RGD.

Radiolabeled cyclic peptides containing the amino acid sequence arginine-glycine-aspartate (RGD peptides) have successfully been used to image the expression of the alpha(v)beta(3) integrin in malignant tumors. However, the alpha(v)beta(3) integrin also plays an important role in angiogenesis induced by chronic inflammatory processes. Therefore, the aim of this study was to evaluate whether radiolabeled RGD peptides may also be used to assess alpha(v)beta(3) expression in inflammatory diseases. We studied a hapten-induced delayed-type hypersensitivity reaction (DTHR) as a model for inflammatory processes, since DTHRs are involved in many human autoimmune disorders. METHODS: The abdominal skin of mice was sensitized by application of 2,4,6-trinitrochlorobenzene (TNCB). One week later, a DTHR was elicited by challenging the right ear with TNCB. Application of TNCB was then repeated every 48 h to induce chronic skin inflammation. Small-animal PET and autoradiography with the alpha(v)beta(3) ligands (18)F-galacto-RGD and (125)I-gluco-RGD were performed at various times after TNCB application. The time course of tracer uptake by the treated ears was compared with histologic skin changes. RESULTS: The first challenge with TNCB caused, within 12 h, an acute inflammatory response with dense dermal infiltrates of polymorphonuclear leukocytes and lymphocytes. However, autoradiography revealed no significant increase in (125)I-gluco-RGD uptake at that time (mean uptake ratio for treated ear to untreated ear, 1.02 +/- 0.1 [SD]). Further challenges with TNCB resulted in chronic skin inflammation with markedly increased small-vessel density in the ear tissue. This was paralleled by a continuous increase in uptake of (125)I-gluco-RGD. After 13 challenges, the uptake ratio had increased to 2.30 +/- 0.27 (P < 0.005 compared with baseline). Enhanced uptake of radiolabeled RGD peptides in chronic inflammation was also demonstrated noninvasively by PET with (18)F-galacto-RGD. Pretreatment of the mice with nonradiolabeled cyclic peptide c(RGDfV) almost completely blocked uptake of (18)F-galacto-RGD by the challenged ear, thus confirming the specificity of tracer uptake. CONCLUSION: Radiolabeled RGD peptides allow a noninvasive assessment of alpha(v)beta(3) expression in inflammatory processes. PET with (18)F-galacto-RGD might become a powerful tool to distinguish between the acute and chronic phases of T cell-mediated immune responses and may represent a new biomarker for disease activity in autoimmune disorders.     

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.     

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.     

<Superscript>18</Superscript>F-labeled mini-PEG spacered RGD dimer (<Superscript>18</Superscript>F-FPRGD2): synthesis and microPET imaging of α<Subscript>v</Subscript>β<Subscript>3</Subscript> integrin expression

Purpose We have previously reported that ¹⁸F-FB-E[c(RGDyK)]₂ (¹⁸F-FRGD2) allows quantitative PET imaging of integrin α_vβ₃ expression. However, the potential clinical translation was hampered by the relatively low radiochemical yield. The goal of this study was to improve the radiolabeling yield, without compromising the tumor targeting efficiency and in vivo kinetics, by incorporating a hydrophilic bifunctional mini-PEG spacer. Methods ¹⁸F-FB-mini-PEG-E[c(RGDyK)]₂ (¹⁸F-FPRGD2) was synthesized by coupling N-succinimidyl-4-¹⁸F-fluorobenzoate (¹⁸F-SFB) with NH₂-mini-PEG-E[c(RGDyK)]₂ (denoted as PRGD2). In vitro receptor binding affinity, metabolic stability, and integrin α_vβ₃ specificity of the new tracer ¹⁸F-FPRGD2 were assessed. The diagnostic value of ¹⁸F-FPRGD2 was evaluated in subcutaneous U87MG glioblastoma xenografted mice and in c-neu transgenic mice by quantitative microPET imaging studies. Results The decay-corrected radiochemical yield based on ¹⁸F-SFB was more than 60% with radiochemical purity of >99%. ¹⁸F-FPRGD2 had high receptor binding affinity, metabolic stability, and integrin α_vβ₃-specific tumor uptake in the U87MG glioma xenograft model comparable to those of ¹⁸F-FRGD2. The kidney uptake was appreciably lower for ¹⁸F-FPRGD2 compared with ¹⁸F-FRGD2 [2.0 ± 0.2%ID/g for ¹⁸F-FPRGD2 vs 3.0 ± 0.2%ID/g for ¹⁸F-FRGD2 at 1 h post injection (p.i.)]. The uptake in all the other organs except the urinary bladder was at background level. ¹⁸F-FPRGD2 also exhibited excellent tumor uptake in c-neu oncomice (3.6 ± 0.1%ID/g at 30 min p.i.). Conclusion Incorporation of a mini-PEG spacer significantly improved the overall radiolabeling yield of ¹⁸F-FPRGD2. ¹⁸F-FPRGD2 also had reduced renal uptake and similar tumor targeting efficacy as compared with ¹⁸F-FRGD2. Further testing and clinical translation of ¹⁸F-FPRGD2 are warranted. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Zhanhong Wu and Zi-Bo Li contributed equally to this work.     

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.     

Phase I trial of the positron-emitting Arg-Gly-Asp (RGD) peptide radioligand 18F-AH111585 in breast cancer patients.

The integrin alpha v beta3 receptor is upregulated on tumor cells and endothelium and plays important roles in angiogenesis and metastasis. Arg-Gly-Asp (RGD) peptide ligands have high affinity for these integrins and can be radiolabeled for PET imaging of angiogenesis or tumor development. We have assessed the safety, stability, and tumor distribution kinetics of a novel radiolabeled RGD-based integrin peptide-polymer conjugate, 18F-AH111585, and its feasibility to detect tumors in metastatic breast cancer patients using PET. METHODS: The biodistribution of 18F-AH111585 was assessed in 18 tumor lesions from 7 patients with metastatic breast cancer by PET, and the PET data were compared with CT results. The metabolic stability of 18F-AH111585 was assessed by chromatography of plasma samples. Regions of interest (ROIs) defined over tumor and normal tissues of the PET images were used to determine the kinetics of radioligand binding in tissues. RESULTS: The radiopharmaceutical and PET procedures were well tolerated in all patients. All 18 tumors detected by CT were visible on the 18F-AH111585 PET images, either as distinct increases in uptake compared with the surrounding normal tissue or, in the case of liver metastases, as regions of deficit uptake because of the high background activity in normal liver tissue. 18F-AH111585 was either homogeneously distributed in the tumors or appeared within the tumor rim, consistent with the pattern of viable peripheral tumor and central necrosis often seen in association with angiogenesis. Increased uptake compared with background (P = 0.002) was demonstrated in metastases in lung, pleura, bone, lymph node, and primary tumor. CONCLUSION: 18F-AH111585 designed to bind the alpha v beta3 integrin is safe, metabolically stable, and retained in tumor tissues and detects breast cancer lesions by PET in most anatomic sites.     

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.     

新型αvβ3和Neuropilin-1双靶点正电子成像探针18F-FAl-NOTA-RGD-ATWLPPR用于脑胶质瘤的PET显像研究

目的构建可靶向αvβ3和血管内皮生长因子受体Neuropilin-1（NRP-1）双受体的正电子成像探针,并验证双靶点融合肽探针较之单靶点探针的优越性。方法采用18F-氟化铝（18F-FAl）配合物的方法实现分子探针的18F标记。在人神经胶质瘤U87MG细胞中,检测αvβ3和NRP-1的表达水平,测定分子探针精氨酸-甘氨酸-天冬氨酸（RGD）-丙氨酸-苏氨酸-色氨酸-亮氨酸-脯氨酸-脯氨酸-精氨酸（ATWLPPR）与αvβ3/NRP-1的受体-配体亲和力。在U87MG荷瘤裸鼠模型中,测定18F标记RGD-ATWLPPR的体内肿瘤micro-PET显像特性,并且与其对应单体进行比较分析。采用方差分析和t检验对结果进行统计学分析。结果αvβ3及NRP-1在U87MG肿瘤细胞、肿瘤组织及肿瘤新生血管中均有较高水平的表达。受体-配体亲和力测定的实验结果显示,18F-FAl-NOTA-RGD-ATWLPPR双靶点融合肽与αvβ3及NRP-1的亲和力并未明显优于其单体,但融合肽在U87MG细胞中的摄取高于相应的单体肽。Micro-PET显像结果显示,融合肽较其单体肽RGD[（4.86±0.48）% ID/g vs.（3.33±0.15）% ID/g,t=10.21,P < 0.05]和ATWLPPR[（4.86±0.48）% ID/g vs.（2.28±0.41）% ID/g,t=32.16,P < 0.05]表现出了更好的显像效果,且融合肽在αvβ3、NRP-1任一受体被未标记“冷”肽阻断的情况下仍能获得肿瘤的阳性显像结果。结论18F-FAl-NOTA-RGD-ATWLPPR可以灵敏地对整合素αvβ3和NRP-1中任何一个受体高表达的肿瘤进行显像,并且较其单体具有更高的肿瘤摄取,但该融合肽的受体-配体亲和力还有待进一步提高。     

Noninvasive imaging of tumor integrin expression using <Superscript>18</Superscript>F-labeled RGD dimer peptide with PEG<Subscript>4</Subscript> linkers

Purpose  Various radiolabeled Arg-Gly-Asp (RGD) peptides have been previously investigated for tumor integrin α_vβ₃ 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 PEG₄ spacers (PEG₄ = 15-amino-4,7,10,13-tetraoxapentadecanoic acid) between the two monomeric RGD motifs and one PEG₄ linker on the glutamate α-amino group (¹⁸F-labeled PEG₄-E[PEG₄-c(RGDfK)]₂, P-PRGD2), as a promising agent for noninvasive imaging of integrin expression in mouse models. Methods  P-PRGD2 was labeled with ¹⁸F via 4-nitrophenyl 2-¹⁸F-fluoropropionate (¹⁸F-FP) prosthetic group. In vitro and in vivo characteristics of the new dimeric RGD peptide tracer ¹⁸F-FP-P-PRGD2 were investigated and compared with those of ¹⁸F-FP-P-RGD2 (¹⁸F-labeled RGD dimer without two PEG₄ spacers between the two RGD motifs). The ability of ¹⁸F-FP-P-PRGD2 to image tumor vascular integrin expression was evaluated in a 4T1 murine breast tumor model. Results  With the insertion of two PEG₄ spacers between the two RGD motifs, ¹⁸F-FP-P-PRGD2 showed enhanced integrin α_vβ₃-binding affinity, increased tumor uptake and tumor-to-nontumor background ratios compared with ¹⁸F-FP-P-RGD2 in U87MG tumors. MicroPET imaging with ¹⁸F-FP-P-PRGD2 revealed high tumor contrast and low background in tumor-bearing nude mice. Biodistribution studies confirmed the in vivo integrin α_vβ₃-binding specificity of ¹⁸F-FP-P-RGD2. ¹⁸F-FP-P-PRGD2 can specifically image integrin α_vβ₃ on the activated endothelial cells of tumor neovasculature. Conclusion   ¹⁸F-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 ¹⁸F-FP-P-PRGD2 a promising agent for PET imaging of tumor angiogenesis and for monitoring the efficacy of antiangiogenic treatment. Electronic supplementary material  The online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

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.