Complementary, selective PET imaging of integrin subtypes α₅β₁ and α_vβ₃ using ⁶⁸Ga-aquibeprin and ⁶⁸Ga-avebetrin

Despite in vivo mapping of integrin α_vβ₃ expression being thoroughly investigated in recent years, its clinical value is still not well defined. For imaging of angiogenesis, the integrin subtype α₅β₁ appears to be a promising target, for which purpose we designed the PET radiopharmaceutical ⁶⁸Ga-aquibeprin. Methods: ⁶⁸Ga-aquibeprin was obtained by click-chemistry (CuAAC) trimerization of a α₅β₁ integrin-binding pseudopeptide on the triazacyclononane-triphosphinate (TRAP) chelator, followed by automated ⁶⁸Ga labeling. Integrin α₅β₁ and α_vβ₃ affinities were determined in enzyme linked immune sorbent assay on immobilized integrins, using fibronectin and vitronectin, respectively, as competitors. M21 (human melanoma)-bearing severe combined immunodeficient mice were used for biodistribution, PET imaging, and determination of in vivo metabolization. The expression of α₅ and β₃ subunits was determined by immunohistochemistry on paraffin sections of M21 tumors. Results: ⁶⁸Ga-aquibeprin shows high selectivity for integrin α₅β₁ (50% inhibition concentration [IC₅₀] 5 0.088 nM) over α_vβ₃ (IC₅₀ 5 620 nM) and a pronounced hydrophilicity (log D 5 -4.2). Severe combined immunodeficient mice xenografted with M21 human melanoma were found suitable for in vivo evaluation, as M21 immunohistochemistry showed not only an endothelial and strong cytoplasmatic expression of the β₃ integrin subunit but also an intense expression of the α₅ integrin subunit particularly in the endothelial cells of intratumoral small vessels. Ex vivo biodistribution (90 min after injection) showed high uptake in M21 tumor (2.42 ± 0.21 percentage injected dose per gram), fast renal excretion, and low background; tumor-to-blood and tumor-to-muscle ratios were 10.6 ± 2.5 and 20.9 ± 2.4, respectively. ⁶⁸Ga-aquibeprin is stable in vivo; no metabolites were detected in mouse urine, blood serum, kidney, and liver homogenates 30 min after injection. PET imaging was performed for ⁶⁸Ga-aquibeprin and the previously described, structurally related c(RGDfK) trimer ⁶⁸Ga-avebetrin, which shows an inverse selectivity for integrin α_vβ₃ (IC₅₀ 5 0.22 nM) over α₅β₁ (IC₅₀ 5 39 nM). In vivo target specificity was proven by cross-competition studies; tumor uptake of either tracer was not affected by the coadministration of 40 nmol (5 mg/kg) of the respective other compound. Conclusion: ⁶⁸Ga-aquibeprin and ⁶⁸Ga-avebetrin are recommendable for complementary mapping of integrins α₅β₁ and α_vβ₃ by PET, allowing for future studies on the role of these integrins in angiogenesis, tumor progression, metastasis, and myocardial infarct healing.