Synthesis of a novel bifunctional chelator AmBaSar based on sarcophagine for peptide conjugation and 64Cu radiolabelling

Cai, Hao; Fissekis, John D.; Conti, Peter S.

doi:10.1039/b902210d

Cited by 78 publications

(86 citation statements)

References 27 publications

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“…34 AmBaSar was activated by EDC and SNHS at pH 4.0 for 30 min (4 °C) with a molar ratio of AmBaSar/EDC/SNHS = 10:9:8. Then, 5-fold molar excess of activated AmBaSar was mixed with the protein polymer in borate buffer (0.1 M, pH 8.5).…”

Section: Materials and Methodsmentioning

confidence: 99%

Multimeric Disintegrin Protein Polymer Fusions That Target Tumor Vasculature

Janib¹,

Gustafson²,

Minea³

et al. 2014

Biomacromolecules

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Recombinant protein therapeutics have increased in number and frequency since the introduction of human insulin, 25 years ago. Presently, proteins and peptides are commonly used in the clinic. However, the incorporation of peptides into clinically approved nanomedicines has been limited. Reasons for this include the challenges of decorating pharmaceutical-grade nanoparticles with proteins by a process that is robust, scalable, and cost-effective. As an alternative to covalent bioconjugation between a protein and nanoparticle, we report that biologically active proteins may themselves mediate the formation of small multimers through steric stabilization by large protein polymers. Unlike multistep purification and bioconjugation, this approach is completed during biosynthesis. As proof-of-principle, the disintegrin protein called vicrostatin (VCN) was fused to an elastin-like polypeptide (A192). A significant fraction of fusion proteins self-assembled into multimers with a hydrodynamic radius of 15.9 nm. The A192-VCN fusion proteins compete specifically for cell-surface integrins on human umbilical vein endothelial cells (HUVECs) and two breast cancer cell lines, MDA-MB-231 and MDA-MB-435. Confocal microscopy revealed that, unlike linear RGD-containing protein polymers, the disintegrin fusion protein undergoes rapid cellular internalization. To explore their potential clinical applications, fusion proteins were characterized using small animal positron emission tomography (microPET). Passive tumor accumulation was observed for control protein polymers; however, the tumor accumulation of A192-VCN was saturable, which is consistent with integrin-mediated binding. The fusion of a protein polymer and disintegrin results in a higher intratumoral contrast compared to free VCN or A192 alone. Given the diversity of disintegrin proteins with specificity for various cell-surface integrins, disintegrin fusions are a new source of biomaterials with potential diagnostic and therapeutic applications.

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Section: Materials and Methodsmentioning

confidence: 99%

Multimeric Disintegrin Protein Polymer Fusions That Target Tumor Vasculature

Janib¹,

Gustafson²,

Minea³

et al. 2014

Biomacromolecules

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“…17,[19][20][21][22][23] Cage amine sarcophagine derived chelators form stable complexes with 64 Cu in vitro and in vivo. [24][25][26][27][28][29][30] In order to develop 64 Cu-labeled GRPr antagonists with improved tumor-to-background ratios we proceeded with the functionalization of the statine-based GRPr antagonist with the sarcophagine (3,6,10,13,16,19-hexaazabicyclo[6.6.6] icosane=Sar) derived chelator, 5-(8-methyl-3,6,10,13,16,19-hexaazabicyclo[6.6.6]icosan-1-ylamino)-5-oxopentanoic acid (MeCOSar), via the spacers PEG4 and PEG2 to obtain LE1 and LE2, respectively, and radiolabel them with 64 Cu. The MeCOSar chelator has excellent pharmacokinetics when coupled to the potent somatostatin receptor targeting peptide Tyr 3 -octreotate and labeled with 64 Cu.…”

Section: Introductionmentioning

confidence: 99%

“…To our knowledge, this is the first time that a GRPr antagonist was functionalized with a Sarderived chelator. Sar derivatives form stable dicationic complexes with Cu 2+ [24][25][26][27][28]. The in vivo stability of 64 Cu-complexes is important, as dissociated 64 Cu 2+ accumulates after binding to albumin in the nuclei or in the mitochondria of non-targeted organs such as the liver 35,36.…”

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confidence: 99%

Copper-64 Labeled Macrobicyclic Sarcophagine Coupled to a GRP Receptor Antagonist Shows Great Promise for PET Imaging of Prostate Cancer

et al. 2015

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The gastrin-releasing peptide receptor (GRPr) is an important molecular target for the visualization and therapy of tumors and can be targeted with radiolabeled bombesin derivatives. The present study aims to develop statine-based bombesin receptor antagonists suitable for labeling with 64Cu for imaging by positron emission tomography (PET). The potent GRPr antagonist D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 was conjugated to the sarcophagine (3,6,10,13,16,19-hexaazabicyclo[6.6.6] icosane=Sar) derivative 5-(8-methyl-3,6,10,13,16,19-hexaaza-bicyclo[6.6.6]icosan-1-ylamino)-5-oxopentanoic acid (MeCOSar) via PEG4 (LE1) and PEG2 (LE2) spacers and radiolabeled with 64Cu2+ with >95% yield and specific activities of about 100 MBq/nmol. Both Cu(II) conjugates have high affinity for GRPr (IC50: natCu-LE1, 1.4±0.1 nM; natCu-LE2, 3.8±0.6 nM). The antagonistic properties of both conjugates were confirmed by Ca2+-flux measurements. Biodistribution studies of Cu-64-LE1 exhibited specific targeting of the tumor (19.6±4.7% IA/g at 1 h p.i.) and GRPr-positive organs. Biodistribution and PET images at 4 and 24 h postinjection showed increasing tumor-to-background ratios with time. This was illustrated by the acquisition of PET images showing high tumor-to-normal tissue contrast. This study demonstrates the high affinity of the MeCOSar-PEGx-bombesin conjugates to GRPr. The stability of 64Cu complexes of MeCOSar, the long half-life of 64Cu, and the suitable biodistribution profile of the 64Cu-labeled peptides lead to PET images of high contrast suitable for potential translation into the clinic.

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“…DOTA and AmBaSar were activated and conjugated to the phage particle using a water-soluble procedure reported earlier 32. For example: 0.75 mg of AmBaSar (1.5 μ mol) dissolved in 20 μ L of water and 0.29 mg of EDC (1.5 μ mol) dissolved in 10 μ L of water were mixed at pH 5.0.…”

Section: Methodsmentioning

confidence: 99%

Trackable and Targeted Phage as Positron Emission Tomography (PET) Agent for Cancer Imaging

Li¹,

Jin²,

Huang³

et al. 2011

Theranostics

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The recent advancement of nanotechnology has provided unprecedented opportunities for the development of nanoparticle enabled technologies for detecting and treating cancer. Here, we reported the construction of a PET trackable organic nanoplatform based on phage particle for targeted tumor imaging. Method: The integrin αvβ3 targeted phage nanoparticle was constructed by expressing RGD peptides on its surface. The target binding affinity of this engineered phage particle was evaluated in vitro. A bifunctional chelator (BFC) 1,4,7,10-tetraazadodecane-N,N',N",N"'-tetraacetic acid (DOTA) or 4-((8-amino-3,6,10,13,16,19-hexaazabicyclo [6.6.6] icosane-1-ylamino) methyl) benzoic acid (AmBaSar) was then conjugated to the phage surface for 64Cu2+ chelation. After 64Cu radiolabeling, microPET imaging was performed in U87MG tumor model and the receptor specificity was confirmed by blocking experiments. Results: The phage-RGD demonstrated target specificity based on ELISA experiment. According to the TEM images, the morphology of the phage was unchanged after the modification with BFCs. The labeling yield was 25 ± 4% for 64Cu-DOTA-phage-RGD and 46 ± 5% for 64Cu-AmBaSar-phage-RGD, respectively. At 1 h time point, 64Cu-DOTA-phage-RGD and 64Cu-AmBaSar-phage-RGD have comparable tumor uptake (~ 8%ID/g). However, 64Cu-AmBaSar-phage-RGD showed significantly higher tumor uptake (13.2 ± 1.5 %ID/g, P<0.05) at late time points compared with 64Cu-DOTA-phage-RGD (10 ± 1.2 %ID/g). 64Cu-AmBaSar-phage-RGD also demonstrated significantly lower liver uptake, which could be attributed to the stability difference between these chelators. There is no significant difference between two tracers regarding the uptake in kidney and muscle at all time points tested. In order to confirm the receptor specificity, blocking experiment was performed. In the RGD blocking experiment, the cold RGD peptide was injected 2 min before the administration of 64Cu-AmBaSar-phage-RGD. Tumor uptake was partially blocked at 1 h time point. Phage-RGD particle was also used as the competitive ligand. In this case, the tumor uptake was significantly reduced and the value was kept at low level consistently. Conclusion: In this report, we constructed a PET trackable nanoplatform based on phage particle and demonstrated the imaging capability of these targeted agents. We also demonstrated that the choice of chelator could have significant impact on imaging results of nano-agents. The method established in this research may be applicable to other receptor/ligand systems for theranostic agent construction, which could have an immediate and profound impact on the field of imaging/therapy and lay the foundation for the construction of next generation cancer specific theranostic agents.

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Synthesis of a novel bifunctional chelator AmBaSar based on sarcophagine for peptide conjugation and 64Cu radiolabelling

Cited by 78 publications

References 27 publications

Multimeric Disintegrin Protein Polymer Fusions That Target Tumor Vasculature

Multimeric Disintegrin Protein Polymer Fusions That Target Tumor Vasculature

Copper-64 Labeled Macrobicyclic Sarcophagine Coupled to a GRP Receptor Antagonist Shows Great Promise for PET Imaging of Prostate Cancer

Trackable and Targeted Phage as Positron Emission Tomography (PET) Agent for Cancer Imaging

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