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

Abstract

Purpose: 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.

Methods: 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 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.

Results: 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 αvβ3-specificity of the peptides.

Conclusion: Our two novel RGD-based radiotracers with optimized pharmacokinetic properties allowed fast, high-contrast PET imaging of tumor-associated integrin αvβ3. These tracers may facilitate the imaging of abdominal malignancies, normally precluded by high background uptake.

Keywords: Angiogenesis; Copper-64 (64Cu); Integrin αvβ3; Molecular imaging; Positron emission tomography (PET); RGD peptide.

Fig. 1

Chemical structures of NOTA-c(RGDfK), NOTA-(PEG)₂-c(RGDfK), and NOTA-PEG₄-SAA₄-c(RGDfK)

Fig. 2

Concentration dependent inhibition of ¹²⁵I-echistatin binding to integrin α_vβ₃ in U87MG cells by c(RGDfK), NOTA-c(RGDfK), NOTA-(PEG)₂-c(RGDfK), or NOTA-PEG₄-SAA₄-c(RGDfK). Solid circles: c(RGDfK) (IC₅₀ = 254 ± 48 nM); solid squares: NOTA-c(RGDfK) (IC₅₀ = 507 ± 62 nM); solid triangles: NOTA-(PEG)₂-c(RGDfK) (IC₅₀ = 444 ± 41 nM); solid rhomboids: NOTA-PEG₄-SAA₄-c(RGDfK) (IC₅₀ = 288 ± 66 nM)

Fig. 3

Dynamic PET-derived time-activity distribution of ⁶⁴Cu-NOTA-c(RGDfK), ⁶⁴Cu-NOTA-(PEG)₂-c(RGDfK), or ⁶⁴Cu-NOTA-PEG₄-SAA₄-c(RGDfK) in blood pool (a), liver (b), kidneys (c), muscle (d), and tumor (e) in nude mice bearing U87MG xenografts, during the first 30 min after injection of 5.5 MBq of the tracers. (f) Time progression of the early tumor-to-muscle ratios for each of the radiolabeled peptides.

Fig. 4

Noninvasive microPET imaging of tumor-associated integrin α_vβ₃ in athymic nude mice bearing U87MG tumors. (a) Representative coronal PET images of planes containing U87MG tumors, at 30 min, 2, and 4 h after intravenous injection of 5.5 MBq of ⁶⁴Cu-NOTA-c(RGDfK), ⁶⁴Cu-NOTA-(PEG)₂-c(RGDfK), ⁶⁴Cu-NOTA-PEG₄-SAA₄-c(RGDfK), or ⁶⁴Cu-NOTA-PEG₄-SAA₄-c(RGDfK) coinjected with an c(RGDyK) (10mg/kg) blocking dose; yellow arrow heads indicate the location of the tumor. (b) Quantitative analysis of the PET images showing the timecourse of the accumulation of the tracers in U87MG tumor, blood pool, liver, kidneys, and muscle. Uptake values are expressed as %ID/g ± SD (n = 3). Bottom right panel describes the tumor-to-muscle ratios attained with each of the radiolabeled peptides.

Fig. 5

(a) Ex vivo biodistribution data of ⁶⁴Cu-NOTA-c(RGDfK), ⁶⁴Cu-NOTA-(PEG)₂-c(RGDfK), and ⁶⁴Cu-NOTA-PEG₄-SAA₄-c(RGDfK) in U87MG bearing nude mice, 4 h after injection. (b) Comparison of the 4 h p.i. biodistribution profile ⁶⁴Cu-NOTA-PEG₄-SAA₄-c(RGDfK) with and without the coinjection of an c(RGDyK) (10mg/kg) blocking dose. Data are represented as %ID/g ± SD (n=3).