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Evaluation of two novel ??Cu-labeled RGD peptide radiotracers for enhanced PET imaging of tumor integrin ?v??.

ABSTRACT: Our goal was to demonstrate that suitably derivatized monomeric RGD peptide-based PET tracers, targeting integrin ?v?3, may offer advantages in image contrast, time for imaging, and low uptake in nontarget tissues.Two cyclic RGDfK derivatives, (PEG)2-c(RGDfK) and PEG4-SAA4-c(RGDfK), were constructed and conjugated to NOTA for (64)Cu labeling. Their integrin ?v?3-binding properties were determined via a competitive cell binding assay. Mice bearing U87MG tumors were intravenously injected with each of the (64)Cu-labeled peptides, and PET scans were acquired during the first 30 min, and 2 and 4 h after injection. Blocking and ex vivo biodistribution studies were carried out to validate the PET data and confirm the specificity of the tracers.The IC50 values of NOTA-(PEG)2-c(RGDfK) and NOTA-PEG4-SAA4-c(RGDfK) were 444?±?41 nM and 288?±?66 nM, respectively. Dynamic PET data of (64)Cu-NOTA-(PEG)2-c(RGDfK) and (64)Cu-NOTA-PEG4-SAA4-c(RGDfK) showed similar circulation t 1/2 and peak tumor uptake of about 4 %ID/g for both tracers. Due to its marked hydrophilicity, (64)Cu-NOTA-PEG4-SAA4-c(RGDfK) provided faster clearance from tumor and normal tissues yet maintained excellent tumor-to-background ratios. Static PET scans at later time-points corroborated the enhanced excretion of the tracer, especially from abdominal organs. Ex vivo biodistribution and receptor blocking studies confirmed the accuracy of the PET data and the integrin ?v?3-specificity of the peptides.Our two novel RGD-based radiotracers with optimized pharmacokinetic properties allowed fast, high-contrast PET imaging of tumor-associated integrin ?v?3. These tracers may facilitate the imaging of abdominal malignancies, normally precluded by high background uptake.

SUBMITTER: Hernandez R

PROVIDER: S-EPMC4591102 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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Evaluation of two novel ⁶⁴Cu-labeled RGD peptide radiotracers for enhanced PET imaging of tumor integrin αvβ₃.

Hernandez Reinier R Czerwinski Andrzej A Chakravarty Rubel R Graves Stephen A SA Yang Yunan Y England Christopher G CG Nickles Robert J RJ Valenzuela Francisco F Cai Weibo W

European journal of nuclear medicine and molecular imaging 20150528 12

<h4>Purpose</h4>Our goal was to demonstrate that suitably derivatized monomeric RGD peptide-based PET tracers, targeting integrin αvβ3, may offer advantages in image contrast, time for imaging, and low uptake in nontarget tissues.<h4>Methods</h4>Two cyclic RGDfK derivatives, (PEG)2-c(RGDfK) and PEG4-SAA4-c(RGDfK), were constructed and conjugated to NOTA for (64)Cu labeling. Their integrin αvβ3-binding properties were determined via a competitive cell binding assay. Mice bearing U87MG tumors were ...[more]

PMID: 26016906

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Project description:Recently, a truncated form of the agouti-related protein (AgRP), a 4-kDa cystine-knot peptide of human origin, was used as a scaffold to engineer mutants that bound to alpha(v)beta(3) integrin with high affinity and specificity. In this study, we evaluated the potential of engineered integrin-binding AgRP peptides for use as cancer imaging agents in living subjects.Engineered AgRP peptides were prepared by solid-phase peptide synthesis and were folded in vitro and purified by reversed-phase high-performance liquid chromatography. Competition assays were used to measure the relative binding affinities of engineered AgRP peptides for integrin receptors expressed on the surface of U87MG glioblastoma cells. The highest-affinity mutant, AgRP clone 7C, was site-specifically conjugated with 1,4,7,10-tetra-azacyclododecane-N,N',N''N'''-tetraacetic acid (DOTA). The resulting bioconjugate, DOTA-AgRP-7C, was radiolabeled with (64)Cu for biodistribution analysis and small-animal PET studies in mice bearing U87MG tumor xenografts. In addition to serum stability, the in vivo metabolic stability of (64)Cu-DOTA-AgRP-7C was assessed after injection and probe recovery from mouse kidney, liver, tumor, and urine.AgRP-7C and DOTA-AgRP-7C bound with high affinity to integrin receptors expressed on U87MG cells (half maximal inhibitory concentration values, 20 +/- 4 and 14 +/- 2 nM, respectively). DOTA-AgRP-7C was labeled with (64)Cu with high radiochemical purity (>99%). In biodistribution and small-animal PET studies, (64)Cu-DOTA-AgRP-7C displayed rapid blood clearance, good tumor uptake and retention (2.70 +/- 0.93 percentage injected dose per gram [%ID/g] and 2.37 +/- 1.04 %ID/g at 2 and 24 h, respectively), and high tumor-to-background tissue ratios. The integrin-binding specificity of (64)Cu-DOTA-AgRP-7C was confirmed in vitro and in vivo by showing that a large molar excess of the unlabeled peptidomimetic c(RGDyK) could block probe binding and tumor uptake. Serum stability and in vivo metabolite assays demonstrated that engineered AgRP peptides are sufficiently stable for in vivo molecular imaging applications.A radiolabeled version of the engineered AgRP peptide 7C showed promise as a PET agent for tumors that express the alpha(v)beta(3) integrin. Collectively, these results validate AgRP-based cystine-knot peptides for use in vivo as molecular imaging agents and provide support for the general use of AgRP as a scaffold to develop targeting peptides, and hence diagnostics, against other tumor receptors.

Project description:PURPOSE:The integrin ?v?6 is overexpressed in a variety of aggressive cancers and serves as a prognosis marker. This study describes the conjugation, radiolabeling, and in vitro and in vivo evaluation of four chelators to determine the best candidate for (64)Cu radiolabeling of A20FMDV2, an ?v?6 targeting peptide. PROCEDURES:Four chelators were conjugated onto PEG28-A20FMDV2 (1): 11-carboxymethyl-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4-methanephosphonic acid (CB-TE1A1P), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), and 4,4'-((3,6,10,13,16,19-hexazazbicyclo[6.6.6]ico-sane-1,8-diylbis(aza-nediyl))bis(methylene)dibenzoic acid (BaBaSar). All peptides were radiolabeled with (64)Cu in ammonium acetate buffer at pH 6 and formulated to pH 7.2 in PBS for use. The radiotracers were evaluated using in vitro cell binding and internalization assays and serum stability assays. In vivo studies conducted include blocking, biodistribution, and small animal PET imaging. Autoradiography and histology were also conducted. RESULTS:All radiotracers were radiolabeled in good radiochemical purity (>95 %) under mild conditions (37-50 °C for 15 min) with high specific activity (0.58-0.60 Ci/?mol). All radiotracers demonstrated ?v?6-directed cell binding (>46 %) with similar internalization levels (>23 %). The radiotracers (64)Cu-CB-TE1A1P-1 and (64)Cu-BaBaSar-1 showed improved specificity for the ?v?6 positive tumor in vivo over (64)Cu-DOTA-1 and (64)Cu-NOTA-1 (+/- tumor uptake ratios-3.82?+/-?0.44, 3.82?±?0.41, 2.58?±?0.58, and 1.29?±?0.14, respectively). Of the four radiotracers, (64)Cu-NOTA-1 exhibited the highest liver uptake (10.83?±?0.1 % ID/g at 4 h). CONCLUSIONS:We have successfully conjugated, radiolabeled, and assessed the four chelates CB-TE1A1P, DOTA, NOTA, and BaBaSar both in vitro and in vivo. However, the data suggests no clear "best candidate" for the (64)Cu-radiolabeling of A20FMDV2, but instead a trade-off between the different properties (e.g., stability, selectivity, pharmacokinetics, etc.) with no obvious effects of the individual chelators.

Dataset Information

Evaluation of two novel ??Cu-labeled RGD peptide radiotracers for enhanced PET imaging of tumor integrin ?v??.

Publications

Evaluation of two novel ⁶⁴Cu-labeled RGD peptide radiotracers for enhanced PET imaging of tumor integrin αvβ₃.

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