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.     

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.     

<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).     

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.     

(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).     

Comparative in vitro and in vivo evaluation of two 64Cu-labeled bombesin analogs in a mouse model of human prostate adenocarcinoma

Bombesin (BBN), an analog of human gastrin-releasing peptide (GRP), binds to the GRP receptor (GRPR) with high affinity and specificity. Overexpression of GRPR has been discovered in mostly androgen-independent human prostate tissues and, thus, provides a potential target for prostate cancer diagnosis and therapy. We have previously demonstrated the feasibility of the positron emission tomography (PET) imaging using 64Cu-1,4,7,10-tetraazadodecane-N,N',N',N'-tetraacetic acid (DOTA)-[Lys3]BBN to detect GRPR-positive prostate cancer. In this study, we compared the receptor affinity, metabolic stability, tumor-targeting efficacy, and pharmacokinetics of a truncated BBN analog 64Cu-DOTA-Aca-BBN(7-14) with 64Cu-DOTA-[Lys3]BBN. Binding of each DOTA conjugate to GRPR on PC-3 and 22Rv1 prostate cancer cells was evaluated with competitive binding assay using 125I-[Tyr4]BBN as radioligand. In vivo pharmacokinetics was determined on male nude mice subcutaneously implanted with PC-3 cells. Dynamic microPET imaging was performed to evaluate the systemic distribution of the tracers. Metabolic stability of the tracers in blood, urine, tumor, liver and kidney was studied using high-performance liquid chromatography. The results showed that 125I-[Tyr4]BBN has a K(d) of 14.8+/-0.4 nM against PC-3 cells, and the receptor concentration on PC-3 cell surface is approximately 2.7+/-0.1 x 10(6) receptors per cell. The 50% inhibitory concentration value for DOTA-Aca-BBN(7-14) is 18.4 +/- 0.2 nM, and that for DOTA-[Lys3]BBN is 2.2 +/- 0.5 nM. DOTA-[Lys3]BBN shows a better tumor contrast and absolute tumor activity accumulation compared to DOTA-Aca-BBN(7-14). Studies on metabolic stability for both tracers on organ homogenates showed that 64Cu-DOTA-[Lys3]BBN is relatively stable. This study demonstrated that both tracers are suitable for targeted PET imaging to detect the expression of GRPR in prostate cancer, while 64Cu-DOTA-[Lys3]BBN may have a better potential for clinical translation.     

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.     

<Superscript>68</Superscript>Ga-labeled NOTA-RGD-BBN peptide for dual integrin and GRPR-targeted tumor imaging

Purpose  Radiolabeled Arg-Gly-Asp (RGD) and bombesin (BBN) peptide analogs have been extensively investigated for the imaging of tumor integrin α_vβ₃ and gastrin-releasing peptide receptor (GRPR) expression, respectively. Recently, we designed and synthesized a RGD-BBN heterodimeric peptide from c(RGDyK) and BBN(7–14) through a glutamate linker. The goal of this study was to investigate the dual receptor-targeting property and tumor diagnostic value of RGD-BBN heterodimeric peptide labeled with generator-eluted ⁶⁸Ga (t_1/2 68 min, β⁺ 89% and EC 11%), ⁶⁸Ga-NOTA-RGD-BBN. Methods  RGD-BBN heterodimer was conjugated with 1,4,7-triazacyclononanetriacetic acid (NOTA) and labeled with ⁶⁸Ga. The dual receptor binding affinity was investigated by a radioligand competition binding assay. The in vitro and in vivo dual receptor targeting of ⁶⁸Ga-NOTA-RGD-BBN was evaluated and compared with that of ⁶⁸Ga-NOTA-RGD and ⁶⁸Ga-NOTA-BBN. Results  NOTA-RGD-BBN had integrin α_vβ₃ and GRPR binding affinities comparable to those of the monomeric RGD and BBN, respectively. The dual receptor targeting property of ⁶⁸Ga-NOTA-RGD-BBN was validated by blocking studies in a PC-3 tumor model. ⁶⁸Ga-NOTA-RGD-BBN showed higher tumor uptake than ⁶⁸Ga-NOTA-RGD and ⁶⁸Ga-NOTA-BBN. ⁶⁸Ga-NOTA-RGD-BBN can also image tumors with either integrin or GRPR expression. Conclusion   ⁶⁸Ga-NOTA-RGD-BBN exhibited dual receptor targeting properties both in vitro and in vivo. The favorable characterizations of ⁶⁸Ga-NOTA-RGD-BBN such as convenient synthesis, high specific activity, and high tumor uptake, warrant its further investigation for clinical cancer imaging. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

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.     

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.     

Dual-function probe for PET and near-infrared fluorescence imaging of tumor vasculature.

To date, the in vivo imaging of quantum dots (QDs) has been mostly qualitative or semiquantitative. The development of a dual-function PET/near-infrared fluorescence (NIRF) probe can allow for accurate assessment of the pharmacokinetics and tumor-targeting efficacy of QDs. METHODS: A QD with an amine-functionalized surface was modified with RGD peptides and 1,4,7,10-tetraazacyclodocecane-N,N',N',N'-tetraacetic acid (DOTA) chelators for integrin alpha(v)beta(3)-targeted PET/NIRF imaging. A cell-binding assay and fluorescence cell staining were performed with U87MG human glioblastoma cells (integrin alpha(v)beta(3)-positive). PET/NIRF imaging, tissue homogenate fluorescence measurement, and immunofluorescence staining were performed with U87MG tumor-bearing mice to quantify the probe uptake in the tumor and major organs. RESULTS: There are about 90 RGD peptides per QD particle, and DOTA-QD-RGD exhibited integrin alpha(v)beta(3)-specific binding in cell cultures. The U87MG tumor uptake of (64)Cu-labeled DOTA-QD was less than 1 percentage injected dose per gram (%ID/g), significantly lower than that of (64)Cu-labeled DOTA-QD-RGD (2.2 +/- 0.3 [mean +/- SD] and 4.0 +/- 1.0 %ID/g at 5 and 18 h after injection, respectively; n = 3). Taking into account all measurements, the liver-, spleen-, and kidney-to-muscle ratios for (64)Cu-labeled DOTA-QD-RGD were about 100:1, 40:1, and 1:1, respectively. On the basis of the PET results, the U87MG tumor-to-muscle ratios for DOTA-QD-RGD and DOTA-QD were about 4:1 and 1:1, respectively. Excellent linear correlation was obtained between the results measured by in vivo PET imaging and those measured by ex vivo NIRF imaging and tissue homogenate fluorescence (r(2) = 0.93). Histologic examination revealed that DOTA-QD-RGD targets primarily the tumor vasculature through an RGD-integrin alpha(v)beta(3) interaction, with little extravasation. CONCLUSION: We quantitatively evaluated the tumor-targeting efficacy of a dual-function QD-based probe with PET and NIRF imaging. This dual-function probe has significantly reduced potential toxicity and overcomes the tissue penetration limitation of optical imaging, allowing for quantitative targeted imaging in deep tissue.     

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.     

Synthesis and biological evaluation of potent alphavbeta3-integrin receptor antagonists

INTRODUCTION: alpha(v)beta(3) Integrin is expressed in sprouting endothelial cells in growing tumors, whereas it is absent in quiescent blood vessels. In addition, various tumor cell types express alpha(v)beta(3) integrin. alpha(v)beta(3) Integrin, a transmembrane heterodimeric protein, binds to the arginine-glycine-aspartic acid (RGD) amino acid sequence of extracellular matrix proteins such as vitronectin and plays a pivotal role in invasion, proliferation and metastasis. Due to the selective expression of alpha(v)beta(3) integrin in tumors, radiolabeled RGD peptides and peptidomimetics are attractive candidates for tumor targeting. METHODS: A cyclic RGD peptide, a peptoid-peptide hybrid, an all-peptoid and a peptidomimetic compound were synthesized, conjugated with 1,4,7,10-tetraazadodecane-N,N',N',N'-tetraacetic acid (DOTA) and radiolabeled with (111)In. Their in vitro and in vivo alpha(v)beta(3)-binding characteristics were determined. RESULTS: IC(50) values were 236 nM for DOTA-E-c(RGDfK), 219 nM for DOTA-peptidomimetic, >10 mM for DOTA-all-peptoid and 9.25 mM for the peptoid-peptide hybrid DOTA-E-c(nRGDfK). (111)In-labeled compounds, except for [(111)In]DOTA-all-peptoid, showed specific uptake in human alpha(v)beta(3)-expressing tumors xenografted in athymic mice. Tumor uptake for [(111)In]DOTA-E-c(RGDfK) was 1.73+/-0.4% ID/g (2 h postinjection) and that of [(111)In]DOTA-peptidomimetic was 2.04+/-0.3% ID/g. Tumor uptake for the peptoid-peptide hybrid [(111)In]DOTA-E-c(nRGDfK) was markedly lower (0.45+/-0.07% ID/g). The all-peptoid [(111)In]DOTA-E-c(nRGnDnFnK) did not show specific uptake in tumors (0.11+/-0.04% ID/g). CONCLUSIONS: The peptidomimetic compound and the cyclic RGD peptide have a high affinity for alpha(v)beta(3) integrin, and these compounds have better tumor-targeting characteristics than the peptoid-peptide hybrid and the all-peptoid.     

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.     

18F-labeled bombesin analogs for targeting GRP receptor-expressing prostate cancer.

The gastrin-releasing peptide receptor (GRPR) is found to be overexpressed in a variety of human tumors. The aim of this study was to develop 18F-labeled bombesin analogs for PET of GRPR expression in prostate cancer xenograft models. METHODS: [Lys3]Bombesin ([Lys3]BBN) and aminocaproic acid-bombesin(7-14) (Aca-BBN(7-14)) were labeled with 18F by coupling the Lys3 amino group and Aca amino group, respectively, with N-succinimidyl-4-18F-fluorobenzoate (18F-SFB) under slightly basic condition (pH 8.5). Receptor-binding affinity of FB-[Lys3]BBN and FB-Aca-BBN(7-14) was tested in PC-3 human prostate carcinoma cells. Internalization and efflux of both radiotracers were also evaluated. Tumor-targeting efficacy and in vivo kinetics of both radiotracers were examined in male athymic nude mice bearing subcutaneous PC-3 tumors by means of biodistribution and dynamic microPET imaging studies. 18F-FB-[Lys3]BBN was also tested for orthotopic PC-3 tumor delineation. Metabolic stability of 18F-FB-[Lys3]BBN was determined in mouse blood, urine, liver, kidney, and tumor homogenates at 1 h after injection. RESULTS: The typical decay-corrected radiochemical yield was about 30%-40% for both tracers, with a total reaction time of 150 +/- 20 min starting from 18F-. 18F-FB-[Lys3]BBN had moderate stability in the blood and PC-3 tumor, whereas it was degraded rapidly in the liver, kidneys, and urine. Both radiotracers exhibited rapid blood clearance. 18F-FB-[Lys3]BBN had predominant renal excretion. 18F-FB-Aca-BBN(7-14) exhibited both hepatobiliary and renal clearance. Dynamic microPET imaging studies revealed that the PC-3 tumor uptake of 18F-FB-[Lys3]BBN in PC-3 tumor was much higher than that of 18F-FB-Aca-BBN(7-14) at all time points examined (P < 0.01). The receptor specificity of 18F-FB-[Lys3]BBN in vivo was demonstrated by effective blocking of tumor uptake in the presence of [Tyr4]BBN. No obvious blockade was found in PC-3 tumor when 18F-FB-Aca-BBN(7-14) was used as radiotracer under the same condition. 18F-FB-[Lys3]BBN was also able to visualize orthotopic PC-3 tumor at early time points after tracer administration, at which time minimal urinary bladder activity was present to interfere with the receptor-mediated tumor uptake. CONCLUSION: This study demonstrates that 18F-FB-[Lys3]BBN and PET are suitable for detecting GRPR-positive prostate cancer in vivo.     

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.     

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.     

99mTc]HYNIC-RGD for imaging integrin alphavbeta3 expression

There has been increasing interest in peptides containing the Arg-Gly-Asp (RGD) sequence for targeting of alpha(v)beta(3) integrins to image angiogenesis. [(18)F]Galacto-RGD has been successfully used for positron emission tomography applications in patients. Here we report on the preclinical characterization of a (99m)Tc-labeled derivative for single-photon emission computed tomography. c(RGDyK) was derivatized with HYNIC at the amino group of the lysine [c(RGDyK(HYNIC)) or HYNIC-RGD]. (99m)Tc labeling was performed using coligands (tricine and EDDA), as well as (99m)Tc(CO)(3)(H(2)O)(3). Radiolabeled peptides were characterized with regard to lipophilicity, protein binding and stability in buffer, serum and tissue homogenates. Integrin receptor activity was determined in internalization assays using alpha(v)beta(3)-receptor-positive M21 and alpha(v)beta(3)-receptor-negative M21L melanoma cells. Biodistribution was evaluated in normal and nude mice bearing M21, M21L and small cell lung tumors. HYNIC-RGD could be labeled at high specific activities using tricine, tricine-trisodium triphenylphosphine 3,3',3'-trisulfonate (TPPTS), tricine-nicotinic acid (NA) or EDDA as coligands. [(99m)Tc]EDDA/HYNIC-RGD, [(99m)Tc]tricine-TPPTS/HYNIC-RGD and [(99m)Tc]tricine-NA/HYNIC-RGD showed protein binding (<5%) considerably lower than [(99m)Tc](CO)(3)/HYNIC-RGD and [(99m)Tc]tricine/HYNIC-RGD. [(99m)Tc]EDDA/HYNIC-RGD revealed high in vitro stability accompanied by low lipophilicity with a log P value of -3.56, comparable to that of [(18)F]Galacto-RGD. In M21 cells for this compound, the highest level of specific and rapid cell uptake (1.25% mg protein(-1)) was determined. In vivo, rapid renal excretion, low blood retention, low liver and muscle uptakes and low intestinal excretion 4 h postinjection were observed. Tumor uptake values were 2.73% ID/g in M21 alpha(v)beta(3)-receptor-positive tumors versus 0.85% ID/g in receptor-negative tumors 1 h postinjection. Small cell lung tumors could be visualized using gamma camera imaging. [(99m)Tc]EDDA/HYNIC-RGD shows encouraging properties to target alpha(v)beta(3) receptors in vivo with high stability and favorable pharmacokinetics. Tumor uptake studies showed specific targeting of alpha(v)beta(3)-receptor-positive tumors with tumor-to-organ ratios comparable to those of [(18)F]Galacto-RGD.     

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.