In vivo biokinetic and metabolic characterization of the 68Ga-labelled α5ß1-selective peptidomimetic FR366.

PURPOSE: Integrins are transmembrane receptors responsible for cell-cell adhesion and cell-extracellular matrix binding and play an important role in angiogenesis and tumour metastasis. For this reason, integrins are increasingly used as targets for molecular imaging. Up to now interest has mostly been focused on the integrin subtype αvß3. However, targeting of other subtypes such as the integrin α5ß1 is also of high interest due to its central role in colonization of metastatic cells, resistance of tumour cells to chemotherapy and ionizing radiation, and tumour aggressiveness. Recently, a highly active antagonist ligand (2,2'-(7-(1-carboxy-4-((6-((3-(4-(((S)-1-carboxy-2-(2-(3-guanidinobenzamido)acetamido)ethyl)carbamoyl)-3,5-dimethylphenoxy)propyl)amino)-6-oxohexyl)amino)-4-oxobutyl)-1,4,7-triazonane-1,4-diyl)diacetic acid, FR366) for the integrin subtype α5ß1 with high selectivity versus αvß3, has been developed and tested successfully in preliminary in vitro and in vivo experiments. Here, we present our results of an investigation of the use of (68)Ga-labelled α5ß1 ligand in PET imaging. METHODS: The free α5ß1 peptidomimetic ligand was functionalized with a spacer (6-aminohexanoic acid) and the bifunctional chelator 1-((1,3-dicarboxy)propyl)-4,7-(carboxymethyl)-1,4,7-triazacyclononane (NODAGA) to yield FR366 and labelled with (68)Ga. To confirm selective in vivo targeting of α5ß1, female BALB/c nude mice xenografted with α5ß1-expressing RKO cells in the right shoulder and α5ß1/αvß3-expressing M21 cells in the left shoulder were subjected to PET/CT scans and biodistribution experiments. Specificity of tracer uptake was proven by blocking studies. Metabolic stability of the injected tracer was measured in urine and in plasma. RESULTS: MicroPET/CT scans with radiolabelled FR366 showed a good tumour-to-normal tissue ratio with low uptake in the liver (0.32 ± 0.14 %ID/g) and good retention of (68)Ga-NODAGA-FR366 in the tumour (0.71 ± 0.20 %ID/g and 0.40 ± 0.12 %ID/g for RKO and M21 tumours, respectively, at 90 min after injection). Biodistribution experiments showed uptake in the α5ß1-expressing RKO tumour of 1.05 ± 0.23 %ID/g at 90 min after injection. Specificity of tracer uptake was demonstrated by injection of 5 mg/kg unlabelled ligand 10 min prior to tracer injection, resulting in a 67 % reduction in uptake in the RKO tumour. The tracer was found to be metabolically stable in urine and plasma 30 min after injection. CONCLUSION: Our results show that PET imaging of α5ß1 expression with the (68)Ga-labelled α5ß1-specific ligand is feasible with good image quality. Thus, FR366 is a promising new tool for investigating the role of α5ß1 in angiogenesis and the influence of this integrin subtype on cancer aggressiveness and metastatic potential.

Multimodal molecular imaging of integrin αvß3 for in vivo detection of pancreatic cancer.

UNLABELLED: Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease. Late detection of then nonresectable or metastasized tumors emphasizes the need for novel imaging approaches. Here, we report on so far nonexploited potentials of αvß3 integrin-targeted molecular imaging technologies for detection of PDAC using genetically engineered mouse models. METHODS: Immunohistochemistry and Western blot were used for characterization of αvß3 expression in murine and human PDAC. We applied IntegriSense 680 fluorescence molecular tomography, intraoperative fluorescence imaging, and (68)Ga-NODAGA-RGD PET for αvß3 integrin molecular in vivo imaging of spontaneous PDAC occurring in Ptf1a(+/Cre);Kras(+/LSL-G12D);p53(LoxP/LoxP) mice. (NODAGA is 1,4,7-triazacyclononane-1,4-bis[acetic acid]-7-[2-glutaric acid] and RGD is arginine-glycine-aspartic acid.) RESULTS: αvß3 integrin is expressed in tumor cells of human and murine PDAC. IntegriSense fluorescence molecular tomography and (68)Ga-NODAGA-RGD PET enabled faithful visualization of PDAC. Furthermore, intraoperative optical imaging with IntegriSense 680 allowed good delineation of tumor borders. CONCLUSION: Imaging approaches targeting αvß3 integrin expand the potential of molecular imaging for identification of αvß3-positive PDAC with potential implications in early detection, fluorescence-guided surgery, and therapy monitoring.

Selective imaging of the angiogenic relevant integrins α5ß1 and αvß3.

Pattern seekers: For the two angiogenic relevant integrins α5ß1 and αvß3, functionalized derivatives of the selective antagonists 1 and 2 could target and discriminate between tumor cells in vivo based on their different integrin patterns and also delay tumor growth in vivo. In addition, the first α5ß1-selective integrin antagonist that enables specific molecular imaging by positron emission tomography was developed.

Be spoilt for choice with radiolabelled RGD peptides: preclinical evaluation of 68Ga-TRAP(RGD)3.

Gallium-68 is rapidly gaining importance, as this generator-produced PET isotope is available independent of on-site cyclotrons, enabling radiopharmaceutical production with comparably simple techniques at low cost. The recently introduced TRAP chelator combines the advantage of straightforward design of multimeric 68Ga-radiopharmaceuticals with very fast and efficient 68Ga-labeling. We synthesized a series of five cyclo(RGDfK) peptide trimers and determined their α(v)ß3 integrin affinities in competition assays on α(v)ß3-expressing M21 human melanoma cells against ¹²5I-echistatin. The compound with highest IC50, Ga-TRAP(RGD)3, showed more than 7-fold higher affinity compared to the monomers F-Galacto-RGD and Ga-NODAGA-c(RGDyK). TRAP(RGD)3 was radiolabeled with 68Ga in a fully automated GMP compliant manner. CD-1 athymic nude mice bearing M21/M21L human melanoma xenografts were used for biodistribution studies, blockade experiments, metabolite studies and PET imaging. 68Ga-TRAP(RGD)3 exhibited high M21 tumor uptake (6.08±0.63% ID/g, 60 min p.i.), was found to be fully stable in vivo, and showed a fast renal clearance. Blockade studies showed that uptake in the tumor, as well as in all other tissues, is highly integrin specific. A comparison of biodistribution and PET data of 68Ga-TRAP(RGD)3 with those of 68Ga-NODAGA-c(RGDyK) and ¹8F-Galacto-RGD showed that the higher affinity of the trimer effects a larger dynamic response of tracer uptake to integrin expression, i.e., enhanced integrin-specific uptake in all tissues. We conclude that 68Ga-TRAP(RGD)3 could allow for imaging of low-level integrin expression in tissues which are not visible with the two competitors. Overall, the study constitutes proof of concept for the favourable in vivo properties of TRAP-based 68Ga radiopharmaceuticals.

Orthogonally protected artificial amino acid as tripod ligand for automated peptide synthesis and labeling with [(99m)Tc(OH(2))(3)(CO)(3)](+).

1,2-Diamino-propionic acid (Dap) is a very strong chelator for the [(99m)Tc(CO)(3)](+) core, yielding small and hydrophilic complexes. We prepared the lysine based Dap derivative l-Lys(Dap) in which the ε-NH(2) group was replaced by the tripod through conjugation to its α-carbon. The synthetic strategy produced an orthogonally protected bifunctional chelator (BFC). The -NH(2) group of the α-amino acid portion is Fmoc- and the -NH(2) of Dap are Boc-protected. Fmoc-l-Lys(Dap(Boc)) was either conjugated to the N- and C-terminus of bombesin BBN(7-14) or integrated into the sequence using solid-phase peptide synthesis (SPPS). We also replaced the native lysine in a cyclic RGD peptide with l-Lys(Dap). For all peptides, quantitative labeling with the [(99m)Tc(CO)(3)](+) core at a 10 µM concentration in PBS buffer (pH = 7.4) was achieved. For comparison, the rhenium homologues were prepared from [Re(OH(2))(3)(CO)(3)](+) and Lys(Dap)-BBN(7-14) or cyclo-(RGDyK(Dap)), respectively. Determination of integrin receptor binding showed low to medium nanomolar affinities for various receptor subtypes. The IC(50) of cyclo-(RGDyK(Dap[Re(CO)(3)])) for α(v)ß(3) is 7.1 nM as compared to 3.1 nM for nonligated RGD derivative. Biodistribution studies in M21 melanoma bearing nude mice showed reasonable α(v)ß(3)-integrin specific tumor uptake. Altogether, orthogonally protected l-Lys(Dap) represents a highly versatile building block for integration in any peptide sequence. Lys(Dap)-precursors allow high-yield (99m)Tc-labeling with [(99m)Tc(OH(2))(3)(CO)(3)](+), forming small and hydrophilic complexes, which in turn leads to peptide radiopharmaceuticals with excellent in vivo characteristics.

68Ga-NODAGA-RGD is a suitable substitute for (18)F-Galacto-RGD and can be produced with high specific activity in a cGMP/GRP compliant automated process.

INTRODUCTION: (18)F-Galacto-cyclo(RGDfK) is a well investigated tracer for imaging of ανß3 expression in vivo, but suffers from the drawback of a time consuming multistep synthesis that can hardly be established under GMP conditions. In this study, we present a direct comparison of the pharmacokinetic properties of this tracer with (68)Ga-NODAGA-cyclo(RGDyK), in order to assess its potential as an alternative for (18)F-Galacto-cyclo(RGDfK). METHODS: (68)Ga labeling of NODAGA-cyclo(RGDyK) was done in full automation using HEPES-buffered eluate of an SnO(2) based (68)Ga-generator. Using M21 (human melanoma) xenografted BALB/c nude mice, biodistribution studies and micro-PET scans were performed for both (18)F-Galacto-cyclo(RGDfK) and (68)Ga-NODAGA-cyclo(RGDyK), and for the latter, in vivo stability was assessed. IC(50) was determined in a displacement assay on M21 cells against (125)I-echistatin. RESULTS: (68)Ga-NODAGA-cyclo(RGDyK) was produced with high specific activity (routinely ca. 500 GBq/µmol) within 15 min. IC(50) values are similar for both substances. Tracer uptake was similar in ανß3 positive tumors (1.45%±0.11% ID/g and 1.35%±0.53% ID/g for (68)Ga-NODAGA-RGD and (18)F-Galacto-RGD, respectively) as well as for all other organs and tissues, with the exception of gall bladder and intestines, where (18)F-Galacto-cyclo(RGDfK) uptake was significantly higher, which can be explained by the higher hydrophilicity of (68)Ga-NODAGA-cyclo(RGDyK) (logP=-4.0 vs. -3.2 for (18)F-Galacto-RGD). Only intact tracer was detected 30 min p.i. in organs and tumor; however, minor amounts of metabolites were found in the urine (6% of total urine activity). CONCLUSION: (68)Ga-labeling of NODAGA-RGD can be performed rapidly and efficiently within 15 min in a GMP compliant process. Similar preclinical results were obtained in comparison with (18)F-Galacto-RGD. Therefore, (68)Ga-NODAGA-cyclo(RGDyK) is a suitable replacement for (18)F-Galacto-cyclo(RGDfK).